RapidSPALM, Protein S-Palmitoylation Detection Kit Application Note Gallery

Application note of RapidSPALM, Protein S-Palmitoylation Detection Kit are listed in Table. Firstly pleasefind ”How to read experimental data of RapidSPALM”.
Table Application of RapidsPALM*Please click title to link each data.
PurposeExampleKit required

Comparison of total S-palmitoylation

Semi-quantitative comparison among mouse tissues

Reaction kit

amounts

Semi-quantitative comparison among cultured cell linesinmultiplesamplesbyfluorometric measurement

Semi-quantitative comparison among broccoli sprout
Semi-quantitative comparison among broccoli tissue

Detection of S-palmitoylation protein

Fluorescent detection of MfTag-labeled proteins in SDS-PAGE gel

Reaction kit

Estimation of S-palmitoyl group number

Estimation of number of S-palmitoyl group in representative marker

Reaction kit

on target proteins by gel shift assay

proteins in mouse whole brain

bands in SDS-PAGE gel by fluorometricimagerComprehensive

purification

ofS-

Purification of S-palmitoylated proteins of four mouse tissues

Reaction kit

palmitoylated proteins by affinity column

Purification of S-palmitoylated proteins of four cultured cells

Purification kit

Purification of S-palmitoylated proteins of broccoli sprout
Purification of S-palmitoylated proteins of broccoli tissue
Identification of S-palmitoylated proteins

Detection of major S-palmitoylated proteins in mouse whole brain

and estimation of S-palm/non-palm ratio

Reaction kit
Purification kitof target proteins by affinity column
Comprehensive analysis

EnrichmentofS-palmitoylated

proteins

by

brain

subcellular

fractionation

Reaction kit
Purification kit

Intracellular distribution of S-palmitoylated proteins in cultured
Neuro2a cells
Drug-induced change of S-palmitoylation in Neuro2a cells
Comparison

withconventional

ABE

Comparison between RapidSPALM and ABE using mouse whole brain

methodReaction kit
Purification kit

Analytic methods for each application are shown below.
➢ Analytical Methods of Fluorometric Assay (Link)➢ Analytical Methods of Gel Shift Assay (Link)➢ Analytic Methods of Affinity Purification (Link)

1

1. How to read experimental data of RapidSPALM
1-1. Control experiment guide
In RapidSPALM experiments, three control experiments for each sample should be prepared. In this webpage,please note hpHA(-)/MfTag(-), hpHA(-)/MfTag(+) and hpHA(+)/MfTag(+) are abbreviated to -/-, -/+, and+/+, respectively. Types of control experiments and their purposes are described in Tables 1 and 2.

FigureStepwise chemical reaction of RapidSPALM

Table 1 Types of control experiment
Experiment name

Abbr.

Reduction

Free-Cys
Blocking

hpHA(-)/MfTag(-)

-/-

✔

✔

hpHA(-)/MfTag(+)

-/+

✔

✔

hpHA(+)/MfTag(+)

+/+

✔

✔

S-palmitoyl
thioestercleavage

MfTag

Desalting-labeling(CMppt)
✔

✔

2

✔

✔

✔

✔

Table-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 onproteins in SDS-PAGE or Western blotting

hpHA(-)/MfTag(+)-/+- Negative control for evaluation of palmitoyl-independent MfTaglabeling- Evaluation of blocking efficiency of non-palmitoyl cysteines

hpHA(+)/MfTag(+)+/+-S-Palmitoylation-specific MfTag-labeled sample

1-2. Selection guide for non-reducing or reducing condition on SDS-PAGE
As MfTag is labeled with thiol of cysteine residue via disulfide bond, MfTag can be easily removed by
reducing reagent (a Kit component). In the Purification kit, Elution buffer will elute intact MfTag-labeledproteins from affinity beads. When SDS-PAGE runs under non-reducing condition, MfTag-labeledproteins show mobility shift on SDS-PAGE depending on it number of MfTag. On the other hand, under
reducing condition MfTags were removed from the proteins and protein bands should be a single band.
You have to select non-reducing condition or reducing condition according to the following
applications (Table 3).

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

3

Table 3 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 column andReducing conditions required

Western blotting4

2. Application Note Gallery◼

Comparison of total S-palmitoylation amounts in multiple samples by fluorometric measurement

Analytical Methods of Fluorometric Assay
Figure 1 shows excitation and emission spectrum of yellow fluorophore (yFL) on the MfTag. Fluorescentintensity at 525 nm excited by 325 nm is recommended. As fluorescent spectrum may be varied by solvent,please use specific buffer in the kit.

Figure 1 Excitation and emission spectrum of yFL on free-MfTag and MfTag-labeled proteinsFor fluorometric assay, measure fluorescent intensity (Ex 325 nm/ Em 525 nm) of three controls, -/-, -/+,and +/+, by each sample.
[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 from oneof the -/+ or +/+ (Eq-1, Figure 2 lower left). After normalization, the ratio of +/+ to -/+ (hpHA(+)/(-), Eq2) 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 multiple samples
For comparison of multiple samples, subtracting -/+ from +/+ indicates “Palmitoylation specific FL
intensity” (Eq-3, Figure 2 lower right).
Palmitoylation specific FL intensity = [+/+] - [-/+]

5(Eq-3)

Figure 2 Analytic methods of fluorometric assay

6

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 (hpHP(+)/(-) =3.5-11.5) was observed. These ratio indicates S-palmitoylspecific 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.
Return to Top

7

Example 2 Semi-quantitative comparison among cultured cells
Samples: Cultured 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 S-palmitoylspecific signal are successfully and preferentially detected.
(B) Semi-quantitative comparison among four cell lines
Among four cultured cell lines tested in this experiment, Neuro2a cells has the largest amount of
total S-palmitoylated proteins.
Return to Top

8Example 3

Semi-quantitative comparison among broccoli sprout

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 S-palimtoylspecific signal are successfully and preferentially detected.
(B) Semi-quantitative comparison among three broccoli sprout tissues
Among three tissues tested in this experiment, roots have the largest amount of total Spalmitoylated proteins.
Return to Top

9Example 4

Semi-quantitative comparison among broccoli tissue

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 S-palmitoylspecific 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 S-palmitoylated
proteins.
Return to Top

10Detection of S-palmitoylation protein bands in SDS-PAGE gel by fluorometric imager
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
SDS-PAGE condition: Non-reducing condition (Keep MfTag-labeling)*Please click here to find selection guide for non-reducing or reducing condition.
Detection: Silver staining and fluorescent imaging (Ex 312 nm/Em>560 nm)

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

11Estimation of S-palmitoyl group number on target proteins by gel shift assay
Analytic Methods of Gel Shift Assay
After conversion of S-palmitoylation to MfTag by Reaction kit, separation of proteins by SDS-PAGE
under non-reducing condition, and detection of individual proteins by Western blotting using antibodies
of interest, you can estimate the number of S-palmitoyl group on the target protein.
[Evaluation]1) Evaluation of S-palmitoylation-specificity
Likely to Figure 1, the protein band of -/- is an original band of the target protein in WB. In theleft case, only the +/+ sample shows that several additional bands and no additional band on -/+is detected. This data indicates that you can successfully prepared -/+ and +/+ samples at least
the indicated protein and the 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 isinsufficient at the least for the indicated protein and you cannot determine the number of Spalmitoylation sites.

Figure 1 Specificity check on gel shift assay
2) Evaluation of each shifted band
As mentioned above, the molecular weight of MfTag is about 5 kDa. If the target protein islabeled with n x MfTags, maximum (n+1) bands (the original band and n x additional bands) willappear. However, in some cases, a certain number of MfTag-labeled bands may not exist in thesample. In these cases, you can consider a number of labeled MfTags on the target protein bymobility distance on the gel and molecular weight plot. Please read the following example.

12

Example: Ras GTPase family member, Hras and Rap2b
Hras and Rap2b are members of Ras small GTPase protein family and reported two Spalmitoylation on the C-terminal region. In the example, the S-palmitoylation status of Hras andRap2b in mouse whole brain was analyzed (Figure 2). For Hras (green), as two additional bands
appeared only in the +/+ sample, the data suggests Hras has two MfTags (=S-palmitoyl group).
For Rap2b (pink) the original band disappeared, and a high molecular weight alternative band
appeared only in the +/+ sample. The number of MfTag on both Hras and Rap2b is calculatedhere.
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 band and place it on the MW-Rf plot above. If theoriginal 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 and 5.7 kDa from the original band (〇), indicating two, one, and zero S-palmitoylated Hras
exist. For Rap2b, the alternative 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 2 Evaluation of MfTag-labeling number of Hras and Rap2b in mouse whole brain tissue*Non-specific bands of antibody

133) Evaluation of the influence of MfTag-labeling on antibody recognition
As MfTag-labeling may affect the antibody-antigen reaction, our gel shift assay should notrecommend comparing each MfTag-labeled band intensity. Figure 3 shows two patterns, theupper indicates MfTag-labeled 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 not detected well, and the relative signal intensity of di-MfTaglabeled protein is dramatically reduced compared to the true value. On the other hand, in thelower case, the antibody interacts with the epitope independently of MfTag-labeling and provides
adequate signal intensity. We strongly recommend the gel shift assay is only for estimation of thenumber of S-palmitoylation, not for estimation of the existing ratio.

Figure 3 Influence of MfTag-labeling on antibody recognition

14

Example Estimation of number of S-palmitoyl group in representative marker proteins in mouse wholebrain
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 referred under each protein name
*Please click here to find selection guide for non-reducing or reducing condition.
Detection method: Western Blotting analysis

Note Asterisk(*) marks indicate non-specific signal of each antibody.

Three types of control experiments -/-, -/+, +/+ 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 against representative seven S-palmitoylated proteins. Allproteins shows 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 mousebrain, these WB data suggest Hras has two, GNAQ has two, Fluotillin-2 has four, Fyn has two, Calnexin
has two and PSD95 has two MfTag (=S-palmitoylated group). As GluN2B is about 150 kDa molecularweight, +/+ specific band shift of GluN2Bwas smear and it was challenging to identify the labeled numberof MfTag.
Return to Top

15

Comprehensive purification of S-palmitoylated proteins by affinity column
Analytic Methods of Affinity Purification
After purification of MfTag-labeled proteins by the Purification Kit, total purified proteins can be visualizedby silver staining following to SDS-PAGE under reducing condition. Furthermore, western blot analysis
allows us to identify individual S-palmitoylated protein. Combination of both reducing condition and nonreducing condition SDS-PAGE/western blotting provides quantitative information about S-palm/nonpalm ratio.
1. Check specificity of each purified protein“Elution” fractions of both -/+ and +/+ are separated by SDS-PAGE under reducing condition forcleaving MfTag and stained by general silver staining. Likely to the left of Figure 1, if only +/+ shows
protein signals, the exchange reaction was successfully achieved. In this condition, each protein band
can be considered as S-palmitoylated proteins. On the other hand, likely to the right of Figure 1, 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-palmitoylated
proteins.

Figure 1 Check specificity of affinity purification

16

2. 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), 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 2). First, see the non-reducing
condition in Figure 2. 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
portion of 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 2 Check purification efficiency and estimate the S-palm ratio

17

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: Reduction condition (Removing MfTag)*Please click here to find selection guide for non-reducing or reducing condition.

A： 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 andthe gel was silver stained. Brain shows the highest amount of purified proteins among four tissues
tested.
B： 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 thegel 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 non-specificprotein.
Return to Top

18

Example 2

Purification of S-palmitoylated proteins of four cultured cells and specificity check

Sample: Cultured Neuro2a, HEK293T, 786-O, Chinese hamster ovary(CHO)
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)*Please click here to find selection guide for non-reducing or reducing condition.

A： 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 thegel was silver stained. Neuro2a cells show a relatively high amount of purified proteins among fourcell lines tested.
B： 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 andthe gel was silver stained. Majority of purified proteins were specifically observed in +/+ samples for
all cell lines.
Return to Top

19

Example 3

Purification of S-palmitoylated proteins of broccoli sprout and specificity check

Samples: Broccoli sprout-derived leaves, stems, and roots
Lysate preparation: Soluble fraction from 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)*Please click here to find selection guide for non-reducing or reducing condition.

A： After purification of MfTag-labeled proteins by the Loop affinity column, Input (10 fold dilution) andElution fraction of +/+ samples were separated by SDS-PAGE under reducing condition and thegel was silver stained. Roots cells show a relatively high amount of purified proteins among threebroccoli sprout tissues tested.
B： After purification of MfTag-labeled proteins by the Loop affinity column, Input (10 fold dilution) andElution fractions of -/-, -/+ and +/+ were separated by SDS-PAGE under reducing condition andthe gel was silver stained. Majority of purified proteins were specifically observed in the +/+ roots
sample.
Return to Top

20

Example 4

Purification of S-palmitoylated proteins of broccoli tissue and specificity check

Samples: Broccoli-derived florets, leaves, and stem
Lysate preparation: Soluble fraction from 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)*Please click here to find selection guide for non-reducing or reducing condition.

A： After purification of MfTag-labeled proteins by the Loop affinity column, Input (10 fold dilution) andElution fraction of +/+ samples were separated by SDS-PAGE under reducing condition and thegel was silver stained. Florets and leaf show relatively high amount of purified proteins than stem.
B： After purification of MfTag-labeled proteins by the Loop affinity column, Input (10 fold dilution) andElution fractions of -/-, -/+ and +/+ of leaf were separated by SDS-PAGE under reducing condition
and the gel was silver stained. Majority of purified proteins were specifically observed in the +/+roots sample. A protein around 50 kDa was detected in the Elution fraction of -/- sample. This
protein binds to the Affinity Beads potentially without MfTag and should be considered a non-specificprotein.
Return to Top

21

Identification of S-palmitoylated proteins and estimation of S-palm/non-palm ratio oftarget proteins by affinity column
Example 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: Reduction condition (Removing MfTag, A, B, C-left)
and Non-reducing condition (Keeping MfTag, C-right)*Please click here to find selection guide for non-reducing or reducing condition.

A： After purification of MfTag-labeled proteins by the Loop affinity column, Input (10 fold dilution), FT(10 fold dilution), 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 indicatedantibodies 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
22

PSDS95 and Calnexin show that only MfTag-labeled bands were detected in the Elution fraction,and the non-labeled band was completely eluted in the FT fraction. This data indicates that MfTaglabeled PSD95 and calnexin were successfully purified in this experiment. Under the condition, thedata of the reduced condition indicates the majority of PSD95 and calnexin are S-palmitoylated
form in adult mouse brain.
Return to Top

23Comprehensive analysis
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 (Removing MfTag)*Please click here to find selection guide for non-reducing or reducing condition.

A： Subcellular fractionation protocol. Whole mouse brain was homogenized in homogenate buffer (50mM 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 P2 fractions. P2 fraction was divided into two batches, and one batch wastreated with 1% TritonX100 Buffer, separated by middle-speed centrifugation (10,000xg) to P2TritonX insoluble fraction (P2-TXinsol) and P2-TritonX soluble fraction (P2-TXsol). Whole brain
homogenate, P2 and P2-TXinsol were solubilized in 1x Basal Buffer and applied to the Reaction
kit.
B： Fluorometric assay. Specificity check (above) and relative comparison (below). These results
24indicate that 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) andElution 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 enrichedin P2 and P2-TXinsol fractions.
Return to Top

25

Example 2

Intracellular distribution of S-palmitoylated proteins in cultured Neuro2a cells

Sample preparation
Rough subcellular fractionation was performed by scheme A. Whole cell lysate, P10k 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*Please click here to find selection guide for non-reducing or reducing condition.

A： 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 (50 mM phosphate (pH 7.4), 150 mM NaCl, 320 mM sucrose, w/o any detergent) withsonication and separated by middle-speed centrifugation (10,000xg, 20 min) to P10k and S10k
fractions. All fractions were done with CMppt, the pellet resolubilized by 1x Basal Buffer, andapplied to the Reaction kit.
B： Fluorometric assay (Specificity check)
C： Total protein amount (left) and estimated S-palmitoylated protein amount (right) calculated from B.
S-palmitoylated proteins are mainly enriched in S10k fraction.
D： After purification of MfTag-labeled proteins, Input (20 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 enriched in S10k fractions.
Return to Top

26

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 homogenizedby homogenization 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-

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
SDS-PAGE condition: Reducing condition (Removing MfTag)and Non-reducing condition (Keeping MfTag)*Please click here to find selection guide for non-reducing or reducing condition.

A： Overview of the experimental protocol
B ： After measurement of fluorescent intensities of each +/+ sample, fluorescent intensities werenormalized by the control experiment. In all cases, after drug treatment the total S-palmitoylation level27

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
SDS-PAGE under both reducing and non-reducing conditions, and gels was silver stained.
Consistent with a fluoremetric assay, purified proteins of NO stress sample were clearly reduced.

Return to Top

28

Reference: Comparison with conventional ABE method
Example Comparison between RapidSPALM and ABE using 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: a half amount of each reacted total proteins
SDS-PAGE condition: Reduction condition (Removing MfTag or biotin)*Please click here to find selection guide for non-reducing or reducing condition.

A Protocol of both methods. Standard ABE method contains 8 times of CMppt (5 times after blocking
and 3 times after biotin-labeling). RapidSPALM contains only 2 times of CMppt.
B Comparison of total protein loss. Silver staining (left) and BCA protein assay (right). RapidSPALM clearly
reduced protein loss and protein aggregation.
C

Comparison of total amount of purified proteins. RapidSPALM dramatically increases purification
amount of proteins. To accurate comparison in silver-staining, 5 fold dilution of RapidSPALM sample
shows same level with ABE on silver staining.
Return to Top

29