PRODUCT INFORMATION
Product Name:

RapidS

PALM, Protein S-Palmitoylation Detection Kit

日本語データシート

Code No. : F017A / F017B※For former lot: 023CC05A, please download datasheet from our website.

Product Name:

RapidS

PALM, Protein S-Palmitoylation Detection Kit

Code No. : F017A<Kit components and Storage condition>
Component Name

Quantity and package

Storage

2x Basal Buffer

1 bottle

RT

Reduction Reagent

1 vial<-20oC

Blocking Reagent A

1 vial<-20oC

Blocking Reagent B

1 vial<-20oC

Cleavage Reagent

1 vial<-20oC

Cleavage Control

1 vial<-20oC

MfTag-labeling Reagent

1 vial<-20oC

Supplement

1 vial<-20oC

Product shelf life : 1 year

Product Name:

RapidS

PALM, Additional Components for Affinity Purification

Code No. : F017B<Kit components and Storage condition>
Component

SizeStore condition

2x Binding Buffer

1 bottle

RT

10x Wash Additive

2 vialsRT

10x Elution Additive

1 vialRT

Empty Column

24 columns

RT

Affinity Beads

4 vials

4oC (DO NOT freeze)

10x Reduction Reagent

1 vial4oC (Before reconstitute)-20oC (After reconstitute)

Product shelf life : 1 year

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PRODUCT INFORMATION

Introduction Part
1. Abstract
RapidS
PALM, Protein S-Palmitoylation Detection Kit is a novel and comprehensive analysis kit for the protein
S-palmitoylation, including S-acylation. S-palmitoyl/acyl groups on proteins are chemically and rapidly
substituted to our unique multifunctional-tag (hereafter MfTag) by multistep chemical reactions. RapidSPALM kit
provides the following information.
1) Quantification of total S-palmitoylation amounts in samples by fluorometric measurement2) Visualization of S-palmitoylation protein bands in SDS-PAGE gel by fluorometric detection3) Estimation of S-palmitoyl group number on target proteins by Western blotting4) Comprehensive purification of S-palmitoylated proteins by affinity column
5) Estimation of S-palmitoylated ratio of target proteins by affinity column and Western blotting
A wide range of samples, including animal tissues, cultured cells, plant tissues, etc., can be applied to the
RapidSPALM kit. Furthermore, RapidSPALM is significantly faster and easier than conventional analytic methods
for protein S-palmitoylation.
2. Overview of the RapidSPALM kit
BioDynamics Laboratory Inc., developed a novel chemical strategy called Rapid Substitution of Protein SAcylation for Multifunctional-tag (RapidSPALM, [rˈæpɪzpάːm]). RapidSPALM can converts the S-palmitoyl group on
proteins to our unique MfTag which can semi-quantitate total amount of S-palmitoylation, purify comprehensively
S-palmitoylated proteins, and count number of S-palmitoyl group. RapidSPALM, Protein S-Palmitoylation
Detection Kit consisting of two kit parts, including a Reaction Kit (Cat. No. #F017A) and Purification Kit (Cat.
No. #F017B) (Figure 1). Firstly, S-palmitoyl groups on proteins in any samples are converted to MfTag by the
Reaction Kit. RapidSPALM’s MfTag contains three functional units, including a yellow fluorophore (yFL), affinity
tag (Hook), and a high molecular weight (~5 kDa) backbone and will label to the cysteine residues, which are
originally S-palmitoylated via a disulfide bond. The Hook group on the MfTag can be specifically and quickly
isolated by a Loop affinity column provided in Purification Kit. After purification, the MfTag is easily cleaved by
reducing reagents.

Figure-1 Overview of RapidSPALM

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PRODUCT INFORMATIONAs mentioned above, RapidSPALM’s MfTag has three functions, 1) yellow fluorophore yFL, 2) high molecularweight structure, and 3) affinity tag Hook, and it provides a total of 5 experimental outputs (Figure 2). 1) yFL isa thermally and photochemically stable fluorescent dye. By yFL group (Ex 300-370 nm/ Em 450-600 nm),amount of MfTag-labeled proteins (=S-palmitoylated proteins) are fluorometrically quantified by any fluorescent
spectrophotometer. Furthermore, MfTag-labeled proteins (=S-palmitoylated proteins) are visualized in-gel by afluorescent imager after SDS-PAGE without any additional staining. 2) The high molecular mass (~5 kDa)
structure of MfTag, will induce a gel shift of labeled proteins on SDS-PAGE. Counting shifted bands indicatesthe number of MfTag (=S-palmitoyl) on proteins by Western blotting. 3) Owing to the Hook group on the MfTagand Loop affinity column-specific interaction, MfTag-labeled proteins (=S-palmitoylated proteins) arecomprehensively isolated by the Loop affinity column, and individual proteins are identified by mass
spectrometry or Western blotting. Furthermore, S-palmitoylated modification ratio can be evaluated by Western
blotting after reducing SDS-PAGE.

Figure-2

Outputs of RapidSPALM

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PRODUCT INFORMATION
3. Principle of RapidSPALM<The principle of Reaction kit>
Our RapidSPALM kit converts S-palmitoyl groups on proteins to a unique MfTag by stepwise chemical reactions(Figure-3) similar to conventional S-palmitoylation detection methods, including Acyl-Biotin Exchange (ABE)and Acyl-PEGyl Exchange Gel Shift Assay (APEGS). Unlike ABE or APEGS, RapidSPALM includes several
original and high-performance reagents with a highly optimized protocol and will reduce non-specific labelingreaction and dramatically shorten each reaction time and omit time-consuming steps. Owing to the RapidSPALM
reagents and protocol, ultra-quick and easy preparation is accomplished.
Step-1 Strong denaturation of proteins
A strong denaturant and heat denature proteins to increase the accessibility of the following reagents.
Step-2 Reduction of disulfide bond
Disulfide bonds are cleaved by a potent reducing reagent.
Step-3 Blocking of free cysteines
Unique and high-performance thiol-specific reagents block free cysteines.
Step-4 Cleavage of S-palmitoyl thioester
S-palmitoyl thioesters are cleaved by our high performance hydroxylamine-derivative (hpHA). Then, hpHA(-)
sample (Tris-treatment) is also prepared as a negative control for the estimation of non-specific MfTaglabeling.
Step-5 MfTag labelingFree-cysteines newly generated from the hpHA cleavage of S-palmitoyl thioester are labeled with MfTag bya cysteine-specific MfTag-labeling reagent. If blocking of non-palmitoyl free-cysteines in Step-3 is insufficient,
MfTag non-specifically binds to proteins in the hpHA(-) sample in S-palmitoylation independent manner.
MfTag(-) sample is also prepared as a completelly negative control.
Step-6 Desalting unreacted reagents by Chloroform/Methanol precipitation (CMppt)
According to reaction procedures above, three control experiments for each sample are required. Hereafter,
each control experiment called hpHA(-)/MfTag(-), hpHA(-)/MfTag(+) and hpHA(+)/MfTag(+) in the manual.

Figure-3

Stepwise chemical reaction of RapidSPALM

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PRODUCT INFORMATION<The principle of Purification kit>
Hook & Loop affinity purification is based on a non-proteinous and small molecular interaction system. Due toa non-protein-based method, all procedures can be performed under completely denatured conditions, and nonspecific binding of applied proteins to affinity column are highly suppressed. The spin-column format provides
an easy procedure, including only the addition of reagent solutions to the column and spinning-down.
4. Advantages of RapidSPALM over conventional ABE method
ABE, the most popular conventional detection method for protein S-palmitoylation, requires over one-day
procedures from sample preparation to affinity purification. Our RapidSPALM kit has ultra-quick procedures andrequires only two hours for the MfTag-labeling reaction and an hour for the purification step. Our RapidSPALM
saves time and labor and provides half-day experiments.

Figure-4 Time course comparison between the conventional ABE and RapidSPALM

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PRODUCT INFORMATION

Protocol Part: Reaction kit (Code : F017A)
Materials Needed but not supplied<Reagents>-

Ultrapure water-

Dimethyl sulfoxide (DMSO)-

Methanol (MeOH)-

Chloroform-

Phosphate buffered saline (PBS)-

BCA protein assay-

2x - 5x Laemmli SDS-PAGE sample buffer (w/o any reduction reagents)<Instruments>- Dry heat block (95oC)- Water bath (37-42oC)- Centrifuge- Vortex mixer- Shaker or Rotator- Probe-type sonicator (Optional)- Water bath sonicator (Optional)- Hand UV lamp (Range: 300-370 nm) (Optional)
Materials Supplied and Storage Condition
The kit contains eight components and will arrive packaged as RT; for best results, remove components andstore as stated below.

Table 0-1 Kit components
Component Name

Quantity and package

Storage

2x Basal Buffer

1 bottle

RT

Reduction Reagent

1 vial<-20oC

Blocking Reagent A

1 vial<-20oC

Blocking Reagent B

1 vial<-20oC

Cleavage Reagent

1 vial<-20oC

Cleavage Control

1 vial<-20oC

MfTag-labeling Reagent

1 vial<-20oC

Supplement

1 vial<-20oCVer. 2.1

PRODUCT INFORMATION
0. Preparation of reagents- Preparation of 1x Basal Buffer
Bring 2x Basal Buffer to room temperature. As 2x Basal Buffer contains a high concentration of salts and adetergent, the salts should be completed dissolved. Warm at 37oC is an option to promote dissolving thesalts. Dilute an appropriate volume of 2x Basal Buffer with the same volume of ultrapure water. The diluted
1x Basal Buffer can be stored at room temperature for the long term. This kit contains 50 mL of 2x Basal
Buffer and enough to prepare 100 mL of 1x Basal Buffer. However, suppose you need a larger volume of
1x Basal Buffer (>100 mL) for preparation of tissue lysates, plant lysates, etc. In that case, you can prepare
an in-house 1x Basal Buffer according to the following buffer composition.
1x Basal Buffer (50 mM sodium phosphate (pH 7.4), 100 mM NaCl, 2% SDS)
‐ Preparation of kit components
The following components are supplied as lyophilized or dried powders/pellets. Bring to room temperatureand dilute the contents with an appropriate volume of solvent mentioned in the list below. If you plan toperform several separate experiments, make aliquots and use fresh for each experiment. Freeze and thaw
cycle is not recommended.
Table 0-2 Reconstitution of lyophilized or dried components
Component

Solvent

VolumeReduction Reagent

Ultrapure water

200 L

Blocking Reagent A

DMSO

100 L

Blocking Reagent B
MfTag-labeling Reagent

DMSO

50 L

Ultrapure water

120 LVer. 2.1

PRODUCT INFORMATION
1. Setting of control experiments
In this kit, as described in Introduction Part, Chapter-3.,we highly recommend using two or three control
experiments for each sample to clarify the specificity of S-palmitoylation. Types of control experiments and their
purposes are described in Table 1-1 and Table 1-2, respectively. In RapidSPALM system, an high-performancehydroxylamine (hpHA) derivative is used as the cleavage reagent of S-palmitoylation/acylation instead of normalhydroxylamine. Please note hpHA(-)/MfTag(-), hpHA(-)/MfTag(+) and hpHA(+)/MfTag(+) are abbreviated to-/-, -/+, and +/+, respectively, in the manual.
Table 1-1 Types of control experiment
Experiment namehpHA(-)/MfTag(-)
hpHA(-)/MfTag(+)
hpHA(+)/MfTag(+)

Abbr.

Reduction

-/-/+
+/+

Free-Cys

S-palmitoyl

Blocking

✔

✔

✔

✔

✔

✔

thioestercleavage

MfTag

Desalting-labeling(CMppt)
✔

✔

✔

✔

✔

✔

Table 1-2 Purpose of each control experiment
Experiment name

Abbr.

hpHA(-)/MfTag(-)-/-

Purpose-Completely negative control-

Evaluation of autofluorescence of each sample-

Evaluation of influence of each chemical reaction in the kit on proteins in
SDS-PAGE or Western blotting

hpHA(-)/MfTag(+)
hpHA(+)/MfTag(+)

-/+
+/+

-

Negative control for evaluation of palmitoyl-independent MfTag-labeling-

Evaluation of blocking efficiency of non-palmitoyl cysteines-

S-Palmitoylation-specific MfTag-labeled sample

In this kit, as S-palmitoyl thioester is cleaved by hpHA, subtraction -/+ from +/+ indicates true S-palmitoylation.
The necessity of each control experiment depends on the output purpose, and a guideline is shown in Table 13. To adequately judge S-palmitoylation-specificity, negative controls in every experiment are highly
recommended.
Table 1-3 Guideline for setting controls
Purpose

-/-

-/+

+/+

Fluorometric assay/detection

✔

✔

✔

Gel shift assay

✔

✔

✔

Affinity purification (Hook & Loop)

*

✔

✔

* For affinity purification, -/- is not needed. If you confirm non-specific binding of proteins to the column, you can set a -/- experiment as anegative control.

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PRODUCT INFORMATION
2. Preparation of sample lysates
Our kit can be used for a wide range of biological samples, including animal tissues, cultured cells, plant tissues,
etc. Before assay, prepare protein lysates with the following guides. In our RapidSPALM kit, all reactions are done
under strong denatured conditions, do not add protease inhibitors. Protease inhibitors may affect the chemical
reaction of the kit; excluding protease inhibitors is highly recommended.

[For cultured cells]
Cultured cells, both floating and adherent cells, are washed with PBS at least twice *1 and lysed with anappropriate volume of 1x Basal Buffer. Prepare over 1 mg/ml total protein lysate. Due to the high concentrationof cellular genomic DNA, the lysate will be highly viscous in the heterogeneous solution. To prepare thehomogeneous solution, carefully shear the DNA lysate by pipetting repeatingly or probe-type sonication*2. Aftersufficient DNA shearing of the lysate, the lysate is centrifuged (10,000 xg, 5 min, RT), and the supernatant iscollected to remove the insoluble fraction, including cell debris or any waste. The protein concentration of thesupernatant is measured by BCA protein assay. Note the 1x Basal Buffer contains a high concentration ofdetergent, Bradford protein assay is not compatible with the Kit. Over 1 mg/ml total protein concentration isdesired for the following reactions*3. Although prepared lysate can be stored at <-20oC, immediate use isrecommended.
*1 If cells are cultured in serum-containing media, wash cells minimum of three times. Contamination of serum
albumin in the cell lysate may cause background signal in this kit.
*2 If less dispersion of lysate with high viscosity may be a reason of low reproducibility.
*3 If the lysate is very low concentration or large volume, find an optional procedure below. You can concentrate
the protein concentration of the lysate.

[For animal tissue]
After mincing, tissues are lysed by appropriate volume of 1x Basal Buffer*4 and homogenized by appropriate
procedures such as homogenizer and sonicator, etc. Sonication procedures are highly recommended topromote extraction of membrane fraction which contains many S-palmitoylated proteins and to prepare
homogeneous cell lysate. After sufficient dispersion of the lysate, the lysate is centrifuged (10,000 xg, 5 min,RT), and the supernatant is collected to remove the insoluble fraction, including cell debris or any waste. The
protein concentration of the supernatant is measured by BCA protein assay. Over 1 mg/ml total protein
concentration is desired for the following reactions. Although prepared lysate can be stored at <-20oC,
immediate use is recommended.
*4 If you need over 5 mL/sample of 1x Basal Buffer, 1x Basal Buffer supplied in the kit will be insufficient for
12 samples. In this case, prepare in-house 1x Basal Buffer described in “0. Preparation of Reagents”.
*5 If the lysate is very low concentration or large volume, find an optional procedure below. You can concentrate
the protein concentration of the lysate.

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PRODUCT INFORMATION[For plant samples]
After grinding plant cells or tissues by appropriate procedures, proteins are extracted with appropriate volumeof 1x Basal Buffer. Insoluble fraction is removed by centrifugation or appropriate filters. Plant samples contain
autofluorescent compounds, including chlorophyll etc., and total protein purification by chloroform/MeOH
precipitation (CMppt) described below is recommended to remove non-protein components. The protein
concentration of the supernatant is measured by BCA protein assay. Over 1 mg/ml total protein concentration
is desired for the following reactions*6. Although prepared lysate can be stored at <-20oC, immediate use isrecommended.
*6 If the lysate is very low concentration or large volume, find an optional procedure below to concentrate theprotein concentration of the lysate.

[Optional procedure: Chloroform/MeOH precipitation for removing nucleotide and lipids]
Chloroform/Methanol precipitation (CMppt) is a phase separation method to prepare protein fractions andremoves nucleotides, lipids, and other biomolecules. CMppt is also useful to concentrate protein and remove
color compounds in samples.
1. Transfer 100 L of sample lysate to a new 1.5 mL tube
2. Add 400 L of MeOH and vigorously mix by vortex
3. Add 100 L of chloroform and vigorously mix by vortex
4. Add 300 L of ultrapure water and vigorously mix by vortex (Solution will turn cloudy)
5. Centrifuge mixture at 10,000xg for 2 min at RT. After centrifugation, phase separation is observed, andproteins precipitate appearing as a thin opaque disk shape between the upper phase and lower phase.
There is no protein pellet at the bottom of the tube at this stage.
6. Carefully remove and discard the upper layer without disturbing the precipitated proteins, it’s not required
to remove the upper phase completely. Leaving a small amount of the upper layer phase is acceptable.
7. Add 300 L of MeOH carefully to the lower phase and gently mix for a homogeneous single phase solution.
Note-1 Carefully mix two phases, do not break the protein pellet.
8. Centrifuge mixture at 10,000xg for 2 min at RT. The protein pellet will be located at the bottom of the tube.
Remove supernatant and discard.
9. Centrifuge tube again at 10,000xg for 2 min at R.T. and remove the supernatant completely.
10. Add appropriate volume of 1x Basal Buffer and dissolve the protein pellet by pipetting or water-bathsonicator. Combine multiple tubes to concentrate the protein concentration.
Note-2 CMppt may induce protein aggregation for some proteins and induce less separatability on SDSPAGE. For our kit, a single CMppt is recommended.

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PRODUCT INFORMATION
3. Assay Protocol of Reaction kit
3-0 Overview of Assay Protocol【1】 Preparation of protein lysate(Reagent component: 1x Basal Buffer)【2】 Denaturation of protein and reduction to cleave disulfide bond – 5 min @95oC(Reagent component: Supplement, Reduction Reagent)【3】

1st Blocking of free-Cys【4】

2nd Blocking of free-Cys– 5 min @RT and additionally 5 min @95 oC(Reagent component: Blocking Reagent A)－ 5 min @RT and additionally 5min @95 oC(Reagent component: Blocking Reagent B)【5】 Cleavage of S-palmitoyl thioester and labeling of MfTag － 40 min@37 oC (~42 oC)(Reagent component: Cleavage Reagent, Cleavage Control, MfTag-labeling Reagent)【6】 Desalting by Chloroform/MeOH precipitation (CMppt) x 2 times － 15 min x 2
Before experiment
Bring all kit components to RT, vortex well, and check solubility. If there is any precipitation, warm the solutions
to 37oC to completely dissolve the salts. Avoid storing components on ice as some components are DMSO
stock solutions; until use, store components at RT.
3-1 Palmitoyl-MfTag Exchange Protocol
1) Dilute total protein lysate prepared in Chapter-2 to 1-2 mg/ml concentration with 1x Basal Buffer.
Note-1 Protein concentration over 2 mg/ml is not recommended, there may be insufficient chemical reaction.
Note-2 Protein concentration less than 1 mg/ml is compatible; however, detection sensitivity after theexchange reaction may be low.
2) Pipet 100 L x number of control of 1-2 mg/ml total protein lysate prepared in step-1). If you use three controls(-/-, -/+ and +/+), 300 L of protein lysate is transferred to a new tube. In the case of two controls (-/+ and+/+), 200 L of protein lysate is transferred to a new tube.
3) Add 1 L x number of control (for three controls; 3 L, for two controls; 2 L) of Supplement and 2.5 L x
number of control (for three controls; 7.5 L, for two controls; 5 L) of Reduction Reagent to protein lysate
and vigorously mix.
Note-3 After the addition of Supplement, BCA assay is not compatible. If BCA assay is required, perform
after step 17).
4) Quick spin-down tubes and incubate tubes for 5 min at 95oC.
5) Add 2 L x number of control (for three controls; 6 L, for two controls; 4 L) of Blocking Reagent A toprotein lysate and vigorously mix.
Note-4 Do not allow cool down to RT. You can add Blocking Reagent A immediately after the heating step.
Be mindful of scalding.
6) Incubate for 5 min with gentle mixing by a rotator or a shaker at RT7) Quick spin-down tubes and incubate for 5 min at 95oC.
8) Add 1 L x number of control (for three controls; 3 L, for two controls; 2 L) of Blocking Reagent B to theprotein lysate and vigorously mix.
Note-5 Do not allow cool down to RT. You can add Blocking Reagent B immediately after the heating step.
Be mindful of scalding.
9) Incubate for 5 min with gentle mixing by a rotator or a shaker at RT

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PRODUCT INFORMATION
10) Quick spin-down tubes and incubate for 5 min at 95oC.
11) Centrifuge the lysate at 10,000xg for 2 min at RT. After centrifugation, the supernatant is divided into 95
L/tubes. Hereafter, -/-, -/+ and +/+ are differently treated.
Note-6 After centrifugation, debris or insoluble pellets of reagent may be present at the bottom of tubes.
Carefully collect supernatant and avoid contamination of precipitation.
12) Add the following reagents to each control and vigorously mix by vortex-/-

Cleavage Control 25 L-/+ Cleavage Control 25 L+/+ Cleavage Reagent 25 L
Note-7 After the addition of Cleavage Reagent for +/+, the solution may become cloudy, which is normal forthe following steps.
13) Add the following reagents to each control and vigorously mix by vortex-/-

ultrapure water 5 L-/+

MfTag-labeling Reagent 5 L+/+ MfTag-labeling Reagent 5 L14) Quick spin-down tubes and incubate for 40 min at 37-42oC. Every 10 minutes, mix the reaction with a vortex.
Note-8 If difficult to incubate at 37-42oC, incubate for 1 hour at RT.
15) Remove unreacted reagents by CMppt
15-1) Add 400 L of MeOH and vigorously mix
15-2) Add 100 L of chloroform and vigorously mix15-3) Add 300 L of ultrapure water and vigorously mix. The solution will turn cloudy.
15-4) Centrifuge at 10,000xg for 2 min at RT. After centrifugation, phase separation is observed, and proteins
precipitate appearing as a thin opaque disk shape between the upper phase and lower phase. There is
no protein pellet at the bottom of the tube at this stage. Carefully remove and discard the upper layer
without disturbing the precipitated proteins, it’s not required to remove the upper phase completely.
Leaving a small amount of the upper layer phase is acceptable.
15-5) Add 300 L of MeOH and mix the two phases by gentle tube inversion. Avoid breaking the protein pellets.
Note-9 If initial protein concentration is very low, protein pellets may not be disc-like precipitation.
15-6) Centrifuge at >10,000xg for 2 min at RT to force the protein pellet to the bottom of the tube andcarefully remove and discard the supernatant.
15-7) Centrifuge at >10,000xg for 30 sec at RT again and remove and discard the supernatant completely.
15-8) Add 100 L of 1x Basal Buffer to the protein pellet; avoid the protein pellet from drying.
15-9) Resuspend the protein pellet completely by pipetting or water-bath sonication.
Note-10 Water-bath sonication is recommended to quickly resuspend the protein pellet.
16) Repeat CMppt again from step-15-1) to 15-7)
Note-11 If you will not perform fluorometric assay or affinity purification assay, 2nd CMppt can be skipped.
17) Hereafter, you need to do a different procedure according to your experimental purpose; follow the flowchart
below.
Note-12 In all cases, the MfTag-labeling can be visualized as emission of yellow fluorescence using a hand
UV lamp in a dark room.

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PRODUCT INFORMATION[Procedure 1]
Resuspend the protein pellet with 110 L of 1x Binding Buffer. After resuspending the protein pellet in 1x
Binding Buffer, affinity purification, fluorometric assay, and gel shift assay can be performed.
Note 1x Binding Buffer is a kit component of Purification Kit.
[Procedure 2]
Resuspend the protein pellet with 50-100 L of 1x Basal Buffer. After doing a fluorometric assay, the sample
can be used for gel shift assay with concentrated Laemmli SDS-PAGE sample buffer (w/o reducing reagents).
[Procedure 3]
Resuspend the protein pellet with any volume of 1x Laemmli SDS-PAGE sample buffer (w/o reducing
reagents) directly.

3-2 Fluorometric assay procedure
For fluorometric assay, measure three controls, -/-, -/+ and +/+, by each sample mentioned in Table 1-3.
1) Heat samples prepared in 3-1 for 2 min at 95oC.
Note-12 This step is essential to measure appropriate fluorescent intensity. As a yellow fluorophore (yFL)group on the MfTag shows great thermal stability, heat step is little effects on yFL decomposition (Figure31B).
2) After cooling to RT, centrifuge samples and collect the supernatant.
3) Measure fluorescent intensity at 525 nm excited by 325 nm with any fluorescent spectrophotometer. Threecontrols, -/-, -/+ and +/+ should be measured. Figure 3-1A shows spectrum and stability of yFL on the
MfTag.
Optional: Fluorescent spectrum from 400 nm to 600 nm exited by 325 nm is helpful to detect appropriate
maximum intensity in each sample and to consider the influence of autofluorescence derived from eachsample.
4) Reuse the samples for performing gel shift assay or affinity purification. When reusing samples, carefullycollect samples and avoid contamination.

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PRODUCT INFORMATION[Important caution]
In RapidSPALM fluorometric assay, there is no internal standard to normalize multi-samples, and fluorescentintensity highly depends on total protein concentration. If you desire to compare the fluorescent intensity ofmulti-samples, normalize total protein concentration by BCA protein assay before measurement of fluorescentintensity. Comparison of multi-samples prepared from different timing is not recommended. The sample group
you desire to compare should be prepared with the same timing.
[Analytical methods]
There are two types of normalization methods of fluorescent intensity.
1) Specificity check of each sample
For checking S-palmitoylation specificity, background fluorescent intensity of -/- is subtracted fromone of the -/+ or +/+ (Eq-1, Figure 3-2 lower left). After normalization, the ratio of +/+ to -/+(hpHA(+)/(-), Eq-2) indicates the S-palmitoylation specificity of the sample preparation. The higher
hpHA(+)/(-) value indicates the higher S-palmitoylation specificity.
Normalized FL intensity = [+/+ or -/+] - [-/-](Eq-1)
Specificity hpHA(+)/(-) = ([+/+] - [-/-])/([-/+] - [-/-])(Eq-2)
2) Comparison of mulple samples
For comparison of multiple samples, subtracting -/+ from +/+ indicates “Palmitoylation specific FL

intensity” (Eq-3, Figure 3-2 lower right).
Palmitoylation specific FL = [+/+]-[-/+](Eq-3)

Figure 3-1 Spectrum and stability of a yellow fluorophore (yFL) on MfTag(A) Excitation and emission spectrum of yFL on free-MfTag and MfTag-labeled proteins.
(B) Thermal stability of yFL on MfTag at 95oC (Ex 325 nm/Em 525 nm).

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PRODUCT INFORMATION

Figure 3-2 Analytic methods of fluorometric assay
3-3. Gel shift assay
For gel shift assay using SDS-PAGE/WB, prepare all three controls (-/-, -/+ and +/+) by each sample.
1) Add appropriate volume of concentrated Laemmli SDS-PAGE sample buffer (from 2x to 5x concentrated,
w/o reducing reagent) to each sample prepared in 3-1 above and heat at 95oC for 2 minutes.
Note Under reducing conditions, MfTag will be removed. Performing SDS-PAGE under non-reducing
conditions is required.
2) Preparation of appropriate concentration of polyacrylamide gel
Table.

Recommended polyacrylamide gel conc.

Molecular weight

Recommended gel conc.

20-30 kDa

13-15%

30-40 kDa

11-13%

40-50 kDa

10-12%

50-70 kDa

8-10%

70-100 kDa

6-8%>100 kDa*<6%*MfTag is about 5 kDa; it may be difficult to estimate the number of MfTag over 100 kDa proteins.
*Separation patterns may be different for each protein. Empirically optimize polyacrylamide gel concentration foryour protein of interest.

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PRODUCT INFORMATION
3) Run SDS-PAGE
4) Transfer to membrane
Both wet and semi-dry transfers are compatible with MfTag-labeled proteins. The transfer conditions will
differ by each protein and require empirically optimizing transfer conditions for your protein of interest.
5) Run Western blotting[Antibody selection guide]
It is very important to select an antibody for gel shift assay with the following guides.
- Highly specific antibody
Highly specific antibodies that detect target proteins with a single band in WB are recommended.
Because MfTag-labelling induces multiple band splits (n+1), where n= MfTag(s) from the original bandit may be hard to detect MfTag-labeled target proteins by low-specific antibodies.
- Polyclonal antibodies are recommended
Because proteins are labeled with a large MW MfTag structure, MfTag may inhibit monoclonal
antibody-target protein interaction if the MfTag-labeling site (=S-palmitoylation site) is located near the
epitope of the monoclonal antibody. Notably, multiple MfTag-labeling near the epitope site ofmonoclonal antibodies induces a dramatic decrease in epitope-antibody interaction. Polyclonal
antibodies with wide-range epitopes are recommended with little influence on the MfTag.
- Trial of several antibodies
When you do the gel shift assay the first time, a trial using several polyclonal antibodies isrecommended.
[Evaluation]1) Evaluation of S-palmitoylation-specificity
Likely to Figure 3-3, the protein band of -/- is an original band of the target protein in WB. In the leftcase, only the +/+ sample shows that several additional bands and no additional band on -/+ is detected.
This data indicates that you successfully prepared -/+ and +/+ samples at least the indicated protein andthe indicated proteins have “two MfTags (=two S-palmitoylation)”. If any additional bands on -/+ appear
likely to the right case, non-specific MfTag-labeling reaction was due to the unblocked free-cysteines.
The right data indicates that blocking of free-cysteine is insufficient at least for the indicated protein andyou cannot determine the number of S-palmitoylation sites.

Figure 3-3 Specificity check on gel shift assay

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2) Evaluation of each shifted band
As mentioned above, the molecular weight of MfTag is about 5 kDa. If the target protein is labeled withn x MfTags, maximum (n+1) bands (the original band and n x additional bands) will appear. However,in some cases, a certain number of MfTag-labeled bands may not exist in the sample. In these cases,
you can consider a number of labeled MfTags on the target protein by mobility distance on the gel andmolecular weight plot. Please read the following example.

Example: Ras GTPase family member, Hras and Rap2b
Hras and Rap2b are members of Ras small GTPase protein family and reported two S-palmitoylation on the
C-terminal region. In the example, the S-palmitoylation status of Hras and Rap2b in mouse whole brain was
analyzed (Figure 3-4). For Hras (green), as two additional bands appeared only in the +/+ sample, the datasuggests Hras has two MfTags (=S-palmitoly group). For Rap2b (pink), the original band disappeared, anda high molecular weight alternative band appeared only in the +/+ sample. The number of MfTag on both
Hras and Rap2b is calculated here.
1) Measure mobility shift (Rf) of each molecular weight (MW) marker proteins and create an MW-Rf plot
and fit curve by the exponential function.
2) Measure Rf for each Hras or Rap2b bands and place it on the MW-Rf plot above
If the original band of +/+ disappears, use original band of -/- or -/+.
3) Calculate the estimated MW of each band shift.
From the MW-Rf plot, for Hras, the second band (●) and the first band (●) were shifted by 10.8 kDa and5.7 kDa from the original band (〇), indicating two, one, and zero S-palmitoylated Hras exist. For Rap2b, thealternative band (●) shifted with 11.0 kDa from the site of the original band (〇). These results indicate
majority of Rap2b has two S-palmitoylation in mouse whole brain.

Figure 3-4 Evaluation of MfTag-labeling number of Hras and Rap2b in mouse whole brain tissue*Non-specific bands of antibody

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PRODUCT INFORMATION[Evaluation of the influence of MfTag-labeling on antibody recognition]
As MfTag-labeling may affect the antibody-antigen reaction, our gel shift assay should not recommend
comparing each MfTag-labeled band intensity. Figure-3-5 shows two patterns, the upper indicates MfTaglabeled sites are overlapped with the antibody epitope, and the lower indicates MfTag-labeled sites are
independent from antibody-epitope interaction. In the upper case, di-MfTag-labeled proteins are notdetected well, and the relative signal intensity of di-MfTag-labeled protein is dramatically reduced comparedto the true value. On the other hand, in the lower case, the antibody interacts with the epitope independentlyof MfTag-labeling and provides adequate signal intensity. We strongly recommend the gel shift assay is
only for estimation of the number of S-palmitoylation, not for estimation of the existing ratio.

Figure 3-5 Influence of MfTag-labeling on antibody recognition

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Protocol Part: Purification kit (Code : F017B)
Materials Required but not supplied<Reagents>-

Ultrapure water-

2x - 5x Laemmli SDS-PAGE sample buffer (without any reducing reagent)<Instrument>- Dry heat block (95oC)- Water bath (37-42oC)- Centrifuge- Hand UV lamp (300-370 nm) (Optional)

Materials Supplied and Storage Condition
The kit contains six components and will arrive packaged as RT; for best results, remove components andstore as stated below.

Table Additional components for Affinity Purification
Component

SizeStore condition

2x Binding Buffer

1 bottle

RT

10x Wash Additive

2 vialsRT

10x Elution Additive

1 vialRT

Empty Column

24 columns

RT

Affinity Beads

4 vials

4oC (DO NOT freeze)

10x Reduction Reagent

1 vial4oC (Before reconstitute)-20oC (After reconstitute)Ver. 2.1

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0. Preparation of reagents
0-1.

Preparation of Binding, Wash, and Elution buffers

In this purification kit, there are three buffers Binding buffer, Wash buffer, and Elution buffer. These buffers
are prepared from 2x Binding buffer and 10x Wash or Elution additive according to the following table.
Salts from the 2x Binding Buffer are easily precipitated at lower temperatures; pre-heat the bottle at 37oC
and confirm the complete dissolving of salts before use. All 1x Buffers should be prepared fresh just before
use.
Preparation of 1x Binding Buffer
x 1 column

x 2 column

x 24 column

2x Binding buffer

200 L400 L

4.8 mL

H2O

200 L400 L

4.8 mLTotal

400 L800 L

9.6 mL

Preparation of 1x Wash Buffer
x 1 column

x 2 column

x 24 column

2x Binding buffer

250 L500 L

6.0 mL

10x Wash additive

50 L

100 L

1.2 mL

H2O

200 L400 L

4.8 mLTotal

500 L

1000 L

12 mL

Preparation of 1x Elution Buffer
x 1 column

x 2 column

x 24 column

2x Binding buffer

75 L

150 L

1,800 L

10x Elution additive

15 L

30 L

360 L

H2O

60 L

120 L

1,440 L

Total

150 L300 L

3,600 L

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1. Purification protocol
1-0. Overview protocol of column purification【1】 Pre-heat samples － @95oC for 2 min and @RT for 5 min【2】 Preparation of spin column － 5 min(Reagents: Empty Column、Affinity Beads、1x Binding Buffer)【3】 Centrifug pre-heated samples － @RT for 2 min【4】 Apply samples to spin column － @RT for >10 min【5】 Wash spin column － ~15 min(Reagents: 1x Binding Buffer, 1x Wash Buffer)【6】 Elution － ~15 min(Reagents: 1x Elution Buffer)
1-1. Protocol for column purification
For column purification of MfTag-labeled proteins, the pair of -/+ and +/+ prepared by our reaction kit is used.
You can optionally use -/- as a complete negative control to check the non-specific purification of non-MfTagged
protein.
1) Protein pellets of -/+ and +/+ samples prepared in the Reaction kit (step 3-1-17) are resuspended with 110
L of 1x Binding Buffer using pipetting or water bath sonicator.
2) After complete resuspension of protein pellets, samples are heated at 95oC for 2 min
3) Samples are cooled down to RT for at least 5 min
Note-1 These samples can be used for fluorometric assay before column purification. Protocol for the
fluorometric assay is described in the Reaction kit manual 3-2.
4) Spin columns are prepared with the following procedures.
4-1) Empty Columns are placed onto either 1.5 mL or 2 mL tubes
4-2) Affinity Beads (50% slurry) are suspended, invert tube several times to resuspend Affinity Beads,
pipet 150 L of Affinity Beads (50% slurry) and transfer to empty column prepared in step-4-1 using awide-bore tip or cut tip.
4-3) Centrifuge the column at 3,000xg for 1 min at RT with open cap and separate Affinity Beads and beads
suspension solution.
4-4) Add 150 L of 1x Binding Buffer to the Affinity Beads and incubate for at least 1 min at RT
Note-2 Affinity Beads Column can be stored for a maximum of a half day. Avoid Affinity Beads from drying.
4-5) Centrifuge the column at 3,000xg for 1 min at RT with open cap and separate Affinity Beads and 1x
Binding Buffer.
4-6) Transfer the equilibrated Affinity Beads Column to a new 1.5 mL or 2 mL tube. The column is read-touse.
5) Centrifuge protein samples prepared in step 3 at 10,000xg, 2 min, RT to remove insoluble debris.
Note-3 If insoluble debris contaminates the column in the next step, background signal may increase. Avoid
contamination of insoluble debris into the column.
6) Pipet 50 L of the centrifuged supernatant sample into the equilibrated Affinity Beads Column and mix thecolumn well by tapping.
7) Incubate for at least 10 min at RT with an open cap.
During incubation, 50 L of the remaining supernatant is transferred to a new 1.5 mL tube. The supernatantis diluted with 50 L of 1x Binding Buffer to prepare 100 L of “Input” Fraction.
8) Centrifuge the column at 3,000xg for 1 min at RT. A new tube exchange is not required.
Note-4 After separation of Affinity Beads and flow-through solution, visually check bead-binding of MfTag-

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labeled proteins as emission of yellow fluorescence with a hand UV lamp. In a successful case, theflow-through solution has little yellow fluorescence, while the affinity beads emit strong fluorescence.
If the flow-through solution shows yellow fluorescence, the bead-binding efficiency of MfTag-labeledproteins is insufficient. In this case, you can increase binding efficiency by re-applying the flowthrough solution to the Affinity Beads and repeating step-6 and step-7.
9) Add 50 L of 1x Wash Buffer to the column and incubate for 2 min with an open cap.
10) Centrifuge the column at 3,000xg for 1 min at RT. A total of 100 L of the flow-through solution is stored as“Flow through (FT)” fraction.
11) Transfer the column to a new 1.5 mL or 2 mL tube.
12) Add 100 L of 1x Wash Buffer to the column, mix the beads by tapping with an open cap, and centrifuge
at 3,000xg for 1 min at RT.
13) Repeat step-12 again and collect a total of 200 L of “Wash-1” fraction.
14) Transfer the column to a new 1.5 mL tube and repeat step-11 and step-12 twice. Collect a total of 200 L of“Wash-2” fraction.
Note-5 If analyzing all wash fractions, exchange tube each time.
15) Transfer the column to a new 1.5 mL tube for the elution sample.
16) Add 25 L of 1x Elution Buffer to the column, incubate for 2 min with an open cap, and centrifuge at
3,000xg for 1 min at RT.
17) Repeat step-15 three times and collect a total of 100 L of “Elution” fraction.
Note-6 After the elution procedure, Affinity Beads are highly denatured and cannot be reused.
Note-7 Elution fraction cannot be applied in a fluorometric assay. The fluorescent intensity of a yellow
fluorophore (yFL) is dramatically decreased in Elution Buffer.
Note-8 As 1x Elution Buffer has a chemical that inhibits BCA assay, Elution fraction cannot be applied to
BCA assay. If checking for protein concentration of the Elution fraction, must perform CMppt, acetone
ppt, or TCA ppt, etc., to remove salts.
18) Add 2x-5x Laemmli SDS-PAGE Sample Buffer to the “Input”, “FT” and “Elution” fractions. Optionallyadd 10x Reduction Reagent to remove MfTag moiety from purified proteins. After adding 10x Reduction
Reagent, the sample is heated at 95oC for 5 min.
Note-9 Elution Buffer will elute intact MfTag-labeled proteins. Under non-reducing conditions, “Elute”
fraction proteins will show a gel shift on SDS-PAGE. Under reducing conditions, MfTags are cleaved
and proteins will show no gel shift on SDS-PAGE.
Note-10 In our protocol, the volumes of “Input”, “FT” and “Elution” are the same 100 L, allowing for thecomparison of the band intensity with WB.
1-2 Analytic methods[Selection guide for non-reducing or reducing condition on SDS-PAGE]
In this kit, Elution Buffer will elute intact MfTag-labeled proteins. When SDS-PAGE runs under nonreducing condition, MfTag-labeled proteins show mobility shift on SDS-PAGE depending on it number of
MfTag. On the other hand, under reducing condition using 10x Reduction Reagent as a kit component,
MfTags were removed from the proteins and protein bands should be a single band (Figure 1-1). After
purification procedures, you have to select non-reducing condition or reducing condition accoding to thefollowing applications (Table 1-1).

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Figure 1-1

Model of two MfTag-labeled proteins under Reduce (-) and Reduce (+) condition

Table 1-1 Selection guide for non-reducing and reducing condition on SDS-PAGE
Condition

Application

Non-reducing condition

Gel shift assay
Fluorescent detection of MfTag-labeled proteins in SDS-PAGE gel

Reducing condition

Comprehensive detection of purified proteins by silver staining
Western blotting detection of purified target proteins

Both non-reducing andEstimation of S-palmitoylated ratio of target proteins by affinity

reducing conditions required

column and Western blotting[Check specificity of each purified protein]“Elution” fractions of both -/+ and +/+ are separated by SDS-PAGE under reducing condition for cleaving
MfTag and stained by general silver staining. Likely to the left of Figure1-2A, if only +/+ shows protein
signals, the exchange reaction was successfully achieved. In this condition, each protein band can beconsidered as S-palmitoylated proteins. On the other hand, likely to the right of Figure1-2A, if -/+ has some
protein bands (white arrows), white arrow-indicated proteins were non-specifically labeled by MfTag
independently S-palmitoylation. These proteins should be excluded from identified S-palmitoylation proteins.

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PRODUCT INFORMATION[Check purification efficiency and estimate ratio of S-palm/non-palm form]
After specific detection of MfTag-labeled proteins (=S-palmitoylated proteins) only observed in the +/+sample mentioned above (Figure 1-2A), an equal volume of “Input”, “FT” and “Elution” fractions of +/+are separated by SDS-PAGE under both reducing condition (w/o MfTag), and non-reducing condition(with MfTag) and proteins of interest are detected by WB (Figure 1-2B). First, see the non-reducing
condition in Figure 1-2B. If affinity purification is successfully done, MfTag-labeled forms (=S-palmitoylated
forms) will disappear in the “FT” fraction (see upper Good case). In this condition, estimating a ratio of
MfTag-labeled and non-labeled forms indicates the ratio of S-palmitoylated and non-palmitoylated forms
under the reducing condition. On the other hand, if affinity purification is insufficiently done, some portionof MfTag-labeled forms remain in the “FT” fraction under non-reducing condition (see lower Bad case).
Estimating a ratio of MfTag-labeled and non-labeled forms cannot be determined under reducing condition.

Figure 1-2 Check specificity (A) and check purification efficiency and estimate the S-palm ratio (B)Ver. 2.1

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Application Note PartNOTE
In the manual, hpHA(-)/MfTag(-), hpHA(-)/MfTag(+) and hpHA(+)/MfTag(+) are hereafter shown as -/-, /+ and +/+, respectively.

Fluorometric assay
Three types of control experiments -/-, -/+ and +/+ for each sample are prepared and measured fluorescentintensity (Ex 325 nm/Em 525 nm) by fluorescent spectrometer. All experiments are analyzed by the two methods
below.
A Specificity test of each sample (-/+ and +/+ are normalized by -/-)B Comparison of different samples (+/+ is normalized by -/+)NOTE Please refere to the Protocol part (Reaction Kit) 3-2. Fluorometric Assay for detail information of eachanalysis method.

Example 1 Semi-quantitative comparison among mouse tissues
Samples: Adult mouse-derived whole brain, kidney, spleen, and liver tissues
Lysate preparation: Whole tissue lysate
Protein amount: 200 g/experimentKit used in this experiment: Reaction Kit(A) Specificity test of each sample
In all samples, high S/N ratio (hpHA(+)/(-) = 3.5-11.5) was observed. These ratio indicates Spalmitoyl-specific signal are successfully and preferentially detected.
(B) Semi-quantitative comparison among four mouse tissues
Among four tissues tested in this experiment, brain tissue has the largest amount of total Spalmitoylated proteins.

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Example 2 Semi-quantitative comparison among cultured cell lines
Samples: Cultured cell lines, Neuro2a, HEK293T, 786-O, and Chinese hamster ovary(CHO)
Lysate preparation: Whole cell lysate
Protein amount: 200 g/experimentKit used in this experiment: Reaction Kit(A) Specificity test of each sample
In all samples, high S/N ratio (hpHA(+)/(-) >11) was observed. These ratio indicates Spalmitoyl-specific signal are successfully and preferentially detected.
(B) Semi-quantitative comparison among four cultured cell lines
Among four cultured cell lines tested in this experiment, Neuro2a cells has the largest amountof total S-palmitoylated proteins.

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Example 3 Semi-quantitative comparison among broccoli sprout tissues
Samples: Broccoli sprout-derived leaves, stems, and roots
Lysate preparation: Soluble fraction from whole tissue lysate
Protein amount: 200 g/experimentKit used in this experiment: Reaction Kit(A) Specificity test of each sample
In all samples, high S/N ratio (hpHA(+)/(-) >3) was observed. These ratio indicates Spalmitoyl-specific signal are successfully and preferentially detected.
(B) Semi-quantitative comparison among three sprout tissues
Among three tissues tested in this experiment, roots have the largest amount of total Spalmitoylated proteins.

Example 4 Semi-quantitative comparison among Broccoli tissues
Samples: Broccoli-derived florets, leaves, stem
Lysate preparation: Soluble fraction from whole tissue lysate
Protein amount: 200 g/experimentKit used in this experiment: Reaction Kit(A) Specificity test of each sample
In all samples, high S/N ratio (hpHA(+)/(-) >3) was observed. These ratio indicates Spalmitoyl-specific signal are successfully and preferentially detected.
(B) Semi-quantitative comparison among three broccoli tissues
Among three tissues tested in this experiment, leaf has the largest amount of total Spalmitoylated proteins.

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In-gel fluorescent detection of labeled proteins
Example Fluorescent detection of MfTag-labeled proteins in SDS-PAGE gel
Sample: Adult mouse-derived whole brain
Lysate preparation: Whole brain lysate
Kit used in this experiment: Reaction kit
Protein amount: 200 g/experiment
SDS-PAGE condition: Non-reducing condition (Keeping MfTag-labeling)
Detection: Silver staining and fluorescent imaging (Ex 312 nm LED/Em >560 nm)

Three types of control experiments -/-, -/+ and +/+ are prepared by Reaction Kit. Each sample was separated
by SDS-PAGE under non-reducing condition to keep MfTag-labeling. Total proteins were detected by silver
staining and MfTag-labeled proteins were visualized by fluorescent imager with 312 nm UV excitation and >560
nm detection. Although there are no change of total protein amount among three experimental samples, majorityof fluorescent signals are specifically detected in +/+.

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Gel shift assay
Example Estimation of number of S-palmitoyl group in representative marker proteins in mouse whole brain
Sample: Adult mouse-derived whole brain
Lysate preparation: Whole brain lysate
Kit used in this experiment: Reaction Kit
Protein amount: 200 g/experiment
SDS-PAGE condition: Non-reducing condition (Keeping MfTag-labeling)Gel concentration is refered under each protein name
Detection method: Western Blotting analysis

Note: * marks in membranes showed non-specific detection of antibodies.

Three types of control experiments -/-, -/+ and +/+ are prepared by Reaction Kit. Each sample was separated
by SDS-PAGE under non-reducing condition to keep MfTag-labeling with appropriate concentration ofpolyacrylamide gel. After SDS-PAGE, proteins on the gel were transferred to PVDF membranes. The
membranes were probed by antibodies agains representative seven S-palmitoylated proteins. All proteinsshows band shifts in only +/+ and no additional bands in -/+. This indicates exchange reaction was
successfully done by Reaction Kit at least seven proteins tested here. In the adult whole mouse brain, these
WB data suggest Hras has two, GNAQ has two, Flotillin-2 has four, Fyn has two, Calnexin has two and
PSD95 has two MfTag (=S-palmitoyl group). As GluN2B is about 150 kDa molecular weight, +/+-specific band
shift of GluN2B was smear and it was challenging to identify the labeled number of MfTag.

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Affinity purification
Example 1 Purification of S-palmitoylated proteins of four mouse tissues and specificity check
Sample: Adult mouse-derived brain, kidney, spleen, liver
Lysate preparation: Whole tissue lysate
Kits used in this experiment: Reaction kit and Purification kit
Protein amount in Reaction kit: 200 g/experimentProtein amount in Purification kit: 100 g/column
SDS-PAGE condition: Reducing condition (Removing MfTag)

A

B

After purification of MfTag-labeled proteins by the Loop affinity column, Input (10 fold dilution) andElution fractions of +/+ samples were separated by SDS-PAGE under reducing condition and the gel
was silver stained. Brain shows the highest amount of purified proteins among four tissues tested.
After purification of MfTag-labeled proteins by the Loop affinity column, Input (20 fold dilution) andElution fractions of -/-, -/+, +/+ were separated by SDS-PAGE under reducing condition and the gel
was silver stained. Purified proteins were specifically observed in +/+ treatment samples for all tissues.
NOTE In the liver sample, a protein around 30 kDa was detected in the Elution fraction of -/- sample.
This protein binds to the Affinity Beads potentially without MfTag and should be considered a nonspecific protein.

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Example 2 Purification of S-palmitoylated proteins of four cultured cells and specificity check
Sample: Cultured Neuro2a, HEK293T, 786-O, CHO cells
Lysate preparation: Whole cell lysate
Kits used in this experiment: Reaction kit and Purification kit
Protein amount in Reaction kit: 200 g/experimentProtein amount in Purification kit: 100 g/column
SDS-PAGE condition: Reducing condition (Removing MfTag)

A

B

After purification of MfTag-labeled proteins by the Loop affinity column, Input (20 fold dilution) andElution fraction of +/+ samples were separated by SDS-PAGE under reducing condition and the gel
was silver stained. Neuro2a cells show a relatively high amount of purified proteins among four cell lines
tested.
After purification of MfTag-labeled proteins by the Loop affinity column, Input (20 fold dilution) andElution fractions of -/-, -/+ and +/+ were separated by SDS-PAGE under reducing condition and thegel was silver stained. Majority of purified proteins were specifically observed in +/+ samples for all celllines.

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Example 3 Detection of major S-palmitoylated proteins in mouse whole brain
Sample: Adult mouse-derived whole brain
Lysate preparation: Whole brain lysate
Kits used in this experiment: Reaction kit and Purification kit
Protein amount in Reaction kit: 200 g/experimentProtein amount in Purification kit: 100 g/column
SDS-PAGE condition: Reducing condition (A, B, C-left) and Non-reducing condition (C-right)

A After purification of MfTag-labeled proteins by the Loop affinity column, Input (10 fold dilution), FT (10 folddilution), and Elution fractions were separated by SDS-PAGE under reducing condition, and the gel was
silver stained. Majority of the purified proteins were specifically observed in +/+.
B Same volume of Input, FT and Elution fractions were applied to SDS-PAGE under reducing condition,
separated proteins were transferred to PVDF membranes, and probed indicated antibodies against seven
representative S-palmitoylated proteins. The WB of Input, FT, and Elution samples ran in the same
membrane; the band intensities can be compared. All seven proteins were specifically detected in +/+ in
Elution fraction. These results indicate this kit successfully detects representative S-palmitoylated proteins.
C Same volume of Input, FT, and Elution fractions of +/+ were applied to SDS-PAGE under both reducing
and non-reducing conditions, separated proteins were transferred to PVDF membranes, and probed
antibodies against PSD95 or calnexin. Under the non-reducing condition, both PSD95 and Calnexin showthat only MfTag-labeled bands were detected in the Elution fraction, and the non-labeled band wascompletely eluted in the FT fraction. This data indicates that MfTag-labeled PSD95 and Calnexin weresuccessfully purified in this experiment. Under the condition, the data of the reduced condition indicatesthe majority of PSD95 and Calnexin are S-palmitoylated form in adult mouse brain.

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Comprehensive application
Example 1 Enrichment of S-palmitoylated proteins by brain subcellular fractionation
Sample preparation
Subcellular fractionation was performed by scheme A. P2 membrane fraction and P2-TritonX100 insoluble
fraction were prepared from whole brain homogenate.
Kits used in this experiment: Reaction kit and Purification kit
Methods: Fluorometric assay, Silver staining of purified proteinsProtein amount in Reaction kit: 100 g/experimentProtein amount in Purification kit: 50 g/column
SDS-PAGE condition: Reducing condition

A

Subcellular fractionation protocol. Whole mouse brain was homogenized in homogenate buffer (50 mM
phosphate (pH 7.4), 150 mM NaCl, 320 mM sucrose, w/o any detergent) with a dounce-type
homogenizer. The whole brain homogenate was initially separated by slow-speed centrifugation(1,000xg) to P1 and S1 fractions. S1 fraction was further separated by middle-speed centrifugation(10,000xg) to P2 and S2 fractions. P2 fraction was divided into two batches, and one batch was treatedwith 1% TritonX100 Buffer, separated by middle-speed centrifugation (10,000xg) to P2-TritonX insoluble
fraction (P2-TXinsol) and P2-TritonX soluble fraction (P2-TXsol). Whole brain homogenate, P2, and P2TXinsol were solubilized in 1x Basal Buffer and applied to the Reaction kit.
B Fluorometric assay. Specificity check (above) and relative comparison (below). These results indicatethat S-palmitoylated proteins were highly accumulated in P2 and P2-TXinsol fractions.
C After purification of MfTag-labeled proteins by the Loop affinity column, Input (5 fold dilution) and Elution
fractions of +/+ were separated by SDS-PAGE under reducing condition, and the gel was silver stained.
Consistent with a fluorometric assay, S-palmitoylated proteins were highly enriched in P2 and P2-TXinsol
fractions.

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Example 2 Intracellular distribution of S-palmitoylated protein in cultured Neuro2a cells
Sample preparation
Rough subcellular fractionation was performed by scheme A. Whole cell lysate, P 10k fraction (mainly
plasma membrane), and S10k fraction (mainly endomembranes and cytosol) were prepared.
Kit: Reaction Kit and Purification Kit
Methods: Fluorometric assay, Silver staining of purified protein
Protein amount in Reaction kit: 100 g/experimentProtein amount in Purification kit: 50 g/column
SDS-PAGE condition: Reducing condition

A

B
C
D

Rough subcellular fractionation protocol. “Whole” was prepared by direct lysis of Neuro2a cell with 1x
Basal Buffer. For the preparation of S10k and P10k, cells were homogenized by homogenate buffer (50mM phosphate (pH 7.4), 150 mM NaCl, 320 mM sucrose, w/o any detergent) with sonication andseparated by middle-speed centrifugation (10,000xg, 20 min) to P10k and S10k fractions. All fractions weredone with CMppt, the pellet resolubilized by 1x Basal Buffer, and applied to the Reaction kit.
Fluorometric assay (Specificity check)
Total protein amount (Left) and estimated S-palmitoylated protein amount (right) calculated from B. Spalmitoylated proteins are mainly enriched in S10k fraction.
After purification of MfTag-labeled proteins, Input (20 fold dilution) and Elution fractions of +/+ wereseparated by SDS-PAGE under reducing condition, and the gel was silver stained. Consistent with afluorometric assay, S-palmitoylated proteins were enriched in S10k fractions.

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Example 3 Drug-induced change of S-palmitoylation in Neuro2a cells
Sample preparation
Neuro2a cells were treated with four drugs for 24 hours. After the treatment, cells were homogenized byhomogenization buffer, and rough plasma membrane fractions (P10k) were separated by the sameprocedure as Example 2.
- Control: Serum-free DMEM
- Protein acyl transferase (PAT) inhibitor: 10 M 2-Bromopalmitate + 10 M Cerulenin in DMEM
- Nitric oxide (NO) stress: 1 mM SNAP DMEM- ER stress: 1 g/ml Tunicamycine in DMEM
- Membrane depolarization: 50 mM KCl in DMEM
Protein amount in Reaction kit: 100 g/experimentProtein amount in Purification kit: 50 g/column
Kits used in this experiment: Reaction kit and Purification kit
Methods: Fluorometric assay, Silver staining of purified proteins

A Overview of the experimental protocol
B After measurement of fluorescent intensities of each +/+ sample, fluorescent intensities were normalized
by the control experiment. In all cases, after drug treatment the total S-palmitoylation level was reduced
and, especially, NO stress dramatically reduced S-palmitoylation level in the PM.
C After purification of MfTag-labeled proteins, Input and Elution samples of +/+ were separated by SDSPAGE under both reducing and non-reducing conditions, and gels was silver stained. Consistent with afluorometric assay, purified proteins of NO stress sample were clearly reduced.

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