ptglab.com

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The Complete Guide To

WESTERN BLOTTING

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Western Blottingptglab.com

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WELCOME
About this guideThis booklet aims to give you a complete guide on how
to perform a Western blot that produces optimal results.
The booklet includes original Western blot lab protocols,
useful technical tips, and troubleshooting.
This guide will take you through the experiment step bystep, enabling you to get the most from your research.

What’s Inside

6–9
Standard Western Blot Protocol
10–11
How To Optimize Your Western Blot
12–13
SDS-PAGE Gel Recipes
14–15
How To Optimize Your Results With Low MW Proteins
16
Tricine Gel Recipe For Low MW Proteins
17
Choosing The Right Lysis Buffer
18–23
Troubleshooting
26–30
Loading Control Antibodies
31
Contact Us4Western Blotting

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  TO YOUR BENCH
Discover the benefits of antibodies from a manufacturer-directcompany for reproducible, reliable data:• Extensively validated in WB, IHC, IF, IP & more.
• Raised against whole protein immunogens.
• Guarantee covers any species/application.
• Antibody specificity demonstrated by siRNA knockdown.

Proteintech® has over 12,000 antibodies in its extensive
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5

6

Western BlottingStandard Western Blot

PROTOCOL
SDS-PAGE

1.

Construct an SDS-PAGE gel according to the molecularweight (MW) of your target protein(s).
See page 12 for SDS-PAGE gel recipes.

2.

Prepare samples in microfuge tubes. Add 4x SDS sample
buffer so the total protein amount is 30–50μg per sample(according to the protein amount measured by Bradford or
BCA protein assay).

3.

Flick microfuge tubes to mix samples, spin them shortly,and then heat to 95–100°C for 5 minutes.

4.

Set up electrophoresis apparatus and immerse in 1x runningbuffer. Remove gel combs and cleanse wells of any residual
stacking gel by pipetting running buffer up and down in eachwell using gel-loading tip (Figure 1).

5.

Load samples and appropriate protein markers onto the gelusing a tip.

6.

Place the lid on the gel tank. Turn on electrophoresis powerpack and set to a low voltage (as the sample runs through thestacking gel), increasing to a higher voltage (e.g., 120V) when
the dye front reaches the separating layer. Stop the gel
running when the dye front migrates to the desired position.

Tip: Tris-tricine gels separate low MW proteins (<20 kDa) better
than Tris-glycine gels. See page 16.

Membrane Transfer

Please note: PVDF membranes (or PSQ membranes
with 0.22μm micropores for targets less than <30 kDa)
are strongly recommended.
1.

Soak membranes in methanol for 30 seconds before
moving to transfer buffer.

2.

Soak the filter papers and sponges in transfer buffer.

3.

Sequentially assemble the transfer constituents according
to the illustration shown on the next page (Figure 2), andensure no bubbles lie between any of the layers. Applysemi-dry or wet transfer systems according to the 		
instructions of the blotting apparatus manufacturer.

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Figure 1

After removing gel combs rinse each well thoroughly with runningbuffer to ensure no remaining bits of acrylamide are blocking them.
Use gel-loading pipette tips to facilitate easy and even loading ofyour protein samples.

Figure 2

A representation of the components of a transfer “sandwich.” Note theorientation of the gel and membrane: the PVDF membrane is situatednearest to the positive electrode. The binding of SDS to proteins resultsin the complex having an overall negative charge. Therefore, the SDSbound proteins travel towards the positive electrode.

8

Immunoblotting

Western Blotting

1.

Following transfer, wash the membrane twice with
distilled water.

2.

Gently mark the bands of the MW ladder on the membraneusing a pencil.

3.

If desired, stain the membrane with commercial Ponceau
red solution for 30 seconds to visualize protein bands,
then wash away Ponceau red solution with generous
amounts of 1x TBST.

4.

Block with 1x TBST containing (2–5%) non-fat dry milk(or 1–5% BSA for the detection of phospho-epitopes)with constant rocking for 1 hour or overnight at 4°C.

5.

Dilute primary antibody in blocking solution with astarting dilution ratio of 1:1000.
(Optimal dilutions should be determined experimentallywith a dilution series.) Incubate the membrane with primary
antibody for 1 hour (or overnight at 4°C) on a benchtop rocker.

6.

Wash membrane three times with 1x TBST for 10
minutes each.

7.

Incubate the membrane with a suitable HRP-conjugatedsecondary antibody (recognizing the host species of the
primary antibody), diluted according to the instructions.
Incubate for 1 hour with constant rocking.

8.

Wash membrane three times with 1x TBST for 10
minutes each.

Tip: Do not let the membrane dry at any stage of the 		blotting process.

Signal Detection

1.

Prepare ECL substrate according to the
manufacturer’s instructions.

2.

Incubate the membrane completely with substrate
for 1–5 minutes (adjust time for more sensitive ECL 		
substrates, e.g., SuperSignal West Femto
Chemiluminescent Substrate [Pierce]).

3.

Expose the membrane to autoradiography film
in a darkroom or read using a chemiluminescenceimaging system.

4.

Line up the developed film in the correct orientation
to the blot and mark the bands of the MW ladder directly
onto the film. It is also advisable to add notes such aslane content, film exposure time, and ECL properties.

Tip: Use multiple exposure lengths to determine the optimal
exposure time. Use fluorescent markers and clip the topright corner of your film as a guide for blot film orientation.

ptglab.com

9Standard Western Blot

PROTOCOL
Buffers Required

1x TBST (1000ml)
20mM Tris-base

2.4 g

150mM NaCl

8.76 g

50mM KCI

3.73 g0.2% Tween-20

2ml

Adjust pH to 7.6.
Add ddH2O to 1000ml
4x SDS Sample Buffer (100ml)
150mM Tris HCl (pH 7.0)

15ml (of a 1M stock)

25% Glycerol

25ml

12% SDS

12 g

0.05% Bromophenol Blue

0.05 g6% ß-mercaptoethanol

6ml

Add ddH2O to 100ml, aliquot, and store at -20ºC.
Wet Transfer Buffer (1000ml)
25mM Tris-base

3.03 g

192mM Glycine

14.4 g

20% Methanol

200ml

Add ddH2O to 1000ml.
Semi-dry Transfer Buffer (1000ml)
48mM Tris-base

5.81 g

39mM Glycine

2.93 g0.0375% SDS

0.375 g20% Methanol

200ml

Add ddH2O to 1000ml

10

Western Blotting

How To Optimize Your

WESTERN BLOT
Obtaining perfect, publication-ready results straightaway is notexactly the norm when performing Western blotting. However,there are steps you can take to refine your blotting experiments
and ensure they get off to the best start possible.
1. SDS-PAGEYour Western blotting experiments start long before you begin
to work with your membrane. If your gel is sloppily made or you
run it at an excessive voltage, your final results will suffer. If you
cast your own gels, take extra care to ensure uniformity in their
makeup. Select the right acrylamide percentage and avoid thatdye-front “smiling” effect by resisting the temptation to run
your gels at high voltages.
Above all, load a uniform amount of protein; the Proteintech
validation lab finds that 0–50ug of protein is a suitable amountfor the detection of most proteins, and usually gives streak-free
and well-separated bands.
2. Membrane
Membrane choice can make a huge difference to the outcomeof your Western blotting experiments. The two main membranetypes are PVDF and nitrocellulose, but there are now several
versions of each on the consumables market.
PVDF tends to be good for lowly expressed proteins, but highbackground and it works better with hydrophilic/polar/charged
target antigens. Nitrocellulose membranes are good for normal
or highly expressed proteins and it works better with hydrophobic/
non-polar antigens.
3. Antibody Concentration
Your next step should be to determine the optimum
working antibody concentration. The rate of binding between
antibody and antigen is affected by their relative concentrations
in solution (among other variables). Often you will need to tweak
the antibody concentration to get better blots. This step is called
antibody titration, and it should be performed every time a new
antibody or new set of experimental conditions is used.

“Often you will need to tweak the antibody
concentration to get better blots.”ptglab.com

11“Remember that milk-based blocking 		
buffers contain such things as endogenous
biotin, glycoproteins, and enzymes that
may interfere with the signal.”

4. Blocking Conditions
Various blocking buffers are available and not all of themwork in every situation. It is important to try several blocking
buffers for each antibody-antigen pair. Remember that milkbased blocking buffers contain such things as endogenous biotin,
glycoproteins, and enzymes that may interfere with the signal.
Milk-based buffers, for example, contain active phosphatasesthat will sabotage your detection of phosphorylated proteins;in these cases, try alternatives such as bovine serum albuminbased blockers.
5. Washing
If the Western blot shows high background signal, revising thewashing steps on the next run-through has proven to be useful.
Increasing washing times and volumes will reduce backgroundsignal along with performing additional buffer changes or using
a stronger detergent (e.g., SDS instead of Tween 20).
6. Detection
The detection stage is integral to obtaining a good Western
blot signal. There are many chemiluminescent reagents andchemiluminescent alternatives out there, such as fluorescentdetection—so there are plenty of options if your current
detection method isn’t meeting lab standards. You can choose
reagents with more sensitivity or those that produce longerlasting signal, for example. The latter choice will increase thereproducibility of blots immeasurably.
A simpler method is to experiment with film-exposure
times. This is probably the optimization step performed mostfrequently, as it is quick and easy. Always expose your blots fora range of individual time points.

12

Western Blotting

SDS-PAGEGEL RECIPES
In order to target proteins with MWs between 20 and 200
kDa, you will need to create a conventional SDS-PAGE gelusing the recipes shown below. The percentage of gel you
require corresponds with the MW of your target protein.

Recipe 1

Separating Gel (mls, total 10ml)
MW of target
protein (kDa)

80-20035-100

25-60

20-40

Gel Percentage

8%

10%

12%

15%

ddH2O

2.1

1.5

0.8

0

30%
Acrylamide

2.7

3.3

4

5

2x Separating
Buffer

5.0

5.0

5.0

5.0

10% APS

0.1

0.1

0.1

0.1

TEMED

0.01

0.01

0.01

0.01

Stacking Gel (mls)
4ml

6ml

8ml

MW of target
protein (kDa)

—

—

—

Gel Percentage

4%

4%

4%

ddH2O

1.4

2.1

2.7

30%
Acrylamide

0.5

0.8

1.1

2x Stacking
Buffer

2.0

3.0

4.0

10% APS

0.04

0.06

0.08

TEMED

0.004

0.006

0.008

ptglab.com

Recipe 213

2x Separating Buffer Recipe (makes 1000ml)
Tris HCl (pH 8.8)

90.8 gSDS

2.0 g

Dissolve compounds thoroughly. Adjust pH slowly to pH 8.8
with concentrated HCl, then add ddH2O to 1000ml.

Recipe 3

2x Stacking Buffer Recipe (makes 1000ml)
Tris HCl (pH 6.8)

30.35 g

SDS

2.0 g

Dissolve compounds thoroughly. Adjust pH slowly to pH 6.8
with concentrated HCl, then add ddH2O to 1000ml.

Recipe 4

1x Running Buffer Recipe (makes 1000ml)
Tris-base1.51 gGlycine

7.5 g

SDS

0.5 g

Dissolve compounds thoroughly, then add ddH2O to 1000ml.

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Antibodies validated with siRNAknockdown to demonstrate specificity.

14

Western Blotting

How To Optimize Your Results With

LOW MW PROTEINSAt extreme ends of the Molecular weight (MW) spectrum,
regular SDS-PAGE and Western blotting techniques sufferfrom limitations: poor separation, signal reduction, or evena total absence of target bands. Low MW proteins (<20 kDa)
are challenging to detect, however several protocol
modifications can be employed when handling these
proteins to improve their retention and resolution.
Use Tricine
In general, glycine gels are ideal for resolving any proteins thatfall within the MW range of 30–250 kDa. However, Acrylamidegels based on a Tris-Tricine buffer system will greatly improveyour chances of “seeing” your target band(s) if you are working
below the 30 kDa range.
You can find a recipe for a 15% Tricine gel on page 16.

17.2kd
14.6kd
10.6kd
8.2kd
6.4kd
2.5kd
A

B

Comparison of Tricine- (A) and glycine-SDS-PAGE (B) separation of
myoglobin fragments. Adapted from Schägger and von Jagow 1987.

Why It Works
The difference in separation capabilities of glycine and tricine
gels is attributed to the differing properties of the glycine andtricine compounds, such as their pK values and ionic mobility.
Tricine is more optimal for the separation of low MW proteinsbecause it “stacks” proteins into more uniform bands. A tricinebased stacking layer shifts the upper stacking limit down to as
low as 30 kDa for the first stack,1 preventing overloading at theinterface between the gel layers.

1 H Schägger and G von Jagow. Tricine-sodium dodecyl sulfate-polyacrylamide
gel electrophoresis for the separation of proteins in the range from 1 to 100
kDa. Anal Biochem. 1987;166(2):368–79.

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Protein Transfer
As well as ensuring the optimum separation of low MW
proteins, you also need to take care at the protein transfer
stage. Low MW proteins are susceptible to “over transfer”– loss of sample due to lack of retention by the transfer
membrane and/or too-rapid transfer.
Membrane Choice and Pore Size
Most labs have a preferred choice of Western blotting
membrane, however PVDF is a better choice in the caseof smaller, low MW proteins, due to its greater capacity tobind proteins. There are various membrane pore sizes onoffer – opt for a smaller pore size to obtain better transfersof your low MW target proteins.

40kd
30kd
20kd
15kd
10kd
5kd

The Proteintech® lab detected 4 kDa sarcolipin protein with anti-sarcoplin
antibody (18395-1-AP; 1:300), following separation on a 15% Tricine gel
and protein transfer to 0.22 μm pore size PVDF membrane.

Transfer Conditions
In addition to membrane choice, factors such as transfer system,
length, temperature, and buffer composition come into play when
dealing with low MW proteins. In the case of small proteins, lessis often more when it comes to transfer length and voltage. How
much you should adjust these by will be down to the brand andmodel of system being used.
Other Tips
Be aware some proteins will escape the dye front of the sample
loading buffer.
You can choose to soak your gel in an SDS-free buffer (or simply
H2O) for 5 minutes prior to setting up your transfer. This helps
to remove SDS, which coats small proteins and protein fragments
in negative charges, increasing their rate of passage through theWestern blot membrane.

16

Western Blotting

Tricine Gel Recipe For

LOW MW PROTEINSFor target proteins with MWs of less than 20 kDa, a tricine
gel system will obtain higher resolution and is highly
recommended. Make three layers of tricine gels as laidout in the following table and diagram. Apply specifictricine gel running buffer to the running system andperform transfer as usual.

RecipeItems

Stacking

Intermediate

Separation

Gel
Percentage

4%

10%

15%

Gel Volume

2 ml

3 ml

6 ml

38% Glycerol

—

—

1.6

ddH2O

1.4

1.2

—

30%
Acrylamide

0.3

0.8

2.7

3.0 M Tris HCl(pH 8.5)

—

1

2.14

1.0 M Tris HCl(pH 6.8)

0.3

—

—

10% SDS

0.02

0.03

0.06

10% APS

0.02

0.03

0.06

TEMED

0.002

0.003

0.003

Stacking Gel
4%Intermediate Gel
10%

Separating Gel
15%

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17

Choosing The Right

LYSIS BUFFER
Preparing
Protein Lysates

Cell lysis is the breaking down of the cell membrane and theseparation of proteins from the non-soluble parts of the cell.
Lysate buffers contain different detergents that help to releasesoluble proteins (Triton-X, Tween, SDS, CHAPS). Dependent on
the location of the protein of interest, a different lysate buffer
is needed to obtain a high yield and purity of the protein.
However, every protein is different and may react differently
with the buffers and detergents. If you don’t get your protein
of interest in solution or you are studying a special protein–protein interaction, you can try different buffers and exchange
the detergents.

General Guidelines

Whole-cell lysate/membrane-bound proteins
The most commonly used buffers are RIPA and NP-40.
RIPA buffer’s harsh properties are best suited for hardto-solubilize proteins.
RIPA: 25mM Tris, HCl (pH 7.6) 150mM NaCl, 1% NP-40, 1%
sodium deoxycholate, 0.1% SDS
NP-40: 50 mM Tris, HCl (pH 8.5) 150 mM NaCl, 1% detergent
Nuclear/mitochondria proteins
RIPA is the preferred choice here. However, fractions
protocols are often used to increase the concentration of thedesired protein.
Cytoplasmic proteins
A Tris-HCl lysis sometimes shows advantages over RIPA buffer.
Optimal conditions should be tested for the protein of interest.
Don’t forget your inhibitors
Denaturation/proteolysis and dephosphorylationen (in caseof phospoproteins) should always be kept to a minimum andadded freshly to the cell lysate (EDTA, sodium orthovanadate,
PSMF, Aprotinin).

18

Western Blotting

TROUBLESHOOTING
Overcome your Western blot difficulties with ourtroubleshooting advice, covering problems suchas weak/no signal, non-specific bands, high signal,and other common issues.

Weak/No Signal

Issues with the primary and/or secondary antibody•

Titrate the antibody to determine optimum concentration.

•

The antibody may have lost activity – perform a dot blot
to determine activity and optimal concentration.

•

Include a positive control (e.g., overexpressed protein,purified protein, positive cell line, etc. Adjust protein
loading accordingly).

•

Change incubation time and temperature (4°C, overnight).

Target protein abundance is too low•

Load more protein per well.

•

Enrich low-abundance proteins by immunoprecipitation,
fractionation, etc.

•

Use appropriate treatment to induce target protein
expression or modification.

•

Ensure sample has not degraded.

•

Include protease inhibitors in the lysis buffer.

•

Use the optimum lysis buffer for the target protein’s
subcellular localization.

•

Check protein loading with an internal loading
control antibody.

Membrane choice•

Select PVDF or NC membranes based on hydrophobicity/hydrophilicity of the target antigen.
–– Check the hydrophobicity/hydrophilicity of theantigen sequence.
–– PVDF membrane works better with hydrophilic/polar/charged target antigens.
–– Nitrocellulose works better with hydrophobic/
non-polar antigens.

ptglab.com

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Blocking buffer issues•

Blocking for too long can mask specific epitopes and prevent
antibody binding.

•

Reduce the percentage of, or remove, the blocking reagent
from the antibody incubation buffers.

•

Switch to using an alternative blocking reagent.

Low molecular weight targets•

Use a Tris-tricine gel for protein targets <20 kDa.
(See page 16 for Tricine gel recipe).

•

Reduce transfer times and/or use smaller pore size
membranes (0.22 μm) for low MW proteins <30 kDa.
(See page 14 for How To Optimize Your Results With Low
MW Proteins).

•

Wet transfer is recommended for small proteins (<10 kDa).

Unsuccessful transfer•

Ensure proper transfer set-up (e.g., no air bubbles trapped
between the gel and the membrane).

•Thicker gels can result in incomplete transfer of highmolecular-weight-proteins.

•

Check the quality of protein transfer with a reversible,
universal protein stain, e.g., Ponceau-S.

•

Wet transfer produces higher-resolution transfers over semidry transfer.

Sodium azide contamination•

The presence of sodium azide inhibits the activity of HRP.

•

Use sodium azide-free buffers.

•

Ensure sufficient washing.

Film exposure too short/
Detection reagent not sensitive enough•Check several exposure times to achieve optimum detection.

•

Try different detection reagent compositions and/or brands.

•

Dilute chemiluminescent reagents in high-purity water.

20

Western BlottingHigh Background

Antibody concentration too high

(Uniform Distribution)•

Use higher antibody dilution.

Insufficient blocking•

Increase the concentration of blocking reagent (e.g., from
5 to 7%).

•

Increase blocking time and/or temperature.

•

Add Tween 20 to the blocking buffer.

•

Include blocking reagent and Tween 20 in the primary
antibody dilution buffer.

Inadequate washing•

Increase washing time and volume.

Dry membrane•

Ensure membrane does not dry out during theimmunoblotting process.

Non-specific binding of secondary antibody•

Perform a secondary antibody-only control experiment (omitthe primary incubation step).

•

Use a pre-adsorbed secondary antibody with reduced crossreactivity to unwanted species.

•

For phosphorylated protein detection, do not use milk-based
buffers such as non-fat milk or casein buffer. (Milk and casein
are phosphoprotein-rich.)

Film exposure too long/Detection reagent too sensitive•

Check different types and dilution of the detection reagent.

ptglab.com

High Background(Non-Specific Bands)

21

The troubleshooting tips for high background (uniform
distribution) can also be applied to scenarios where
non-specific but distinct bands appear on the Western
blot membrane.
The following should also be considered:
Target protein abundance is lower than threshold of nonspecific binding•

Load more protein per well at SDS-PAGE.

•

Enrich low-abundance proteins by immunoprecipitation,
fractionation, etc.

Sample degradation•

Prepare fresh lysates each time•

Use freshly prepared sample kept on ice up until
the addition of sample buffer and immediate heating
to 95°C for 5–10 minutes.

•

Tissue extracts tend to produce more non-specificbands and degradation products. Use fresh, sonicated,and clarified tissue extracts.

•

Always include protease inhibitors (and phosphataseinhibitors for the detection of phosphorylated targets).

•

Ensure sample has not degraded.

Interference from other isoforms•

Check for the presence of known isoforms in the literature
or at uniprot.org.

•

Use an isoform-specific antibody.

Inefficient SDS-PAGE separation•

Change the gel percentage to suit the target protein’s MW.

•

Lower percentage Tris-Glycine gels should be used for larger
proteins, or use Tris-Acetate-based gels and buffers.
(See page 12 for our SDS-PAGE gel recipes).

•

Higher percentage Tris-Glycine gels (up to 15%) should be
used for smaller proteins (<20 kDa) or use Tris-Tricine gels.
(See page 16 for our Tricine gel recipe).

Presence of post-translational modifications•

Know your protein of interest, band sizes can shift due toglycosylation, phosphorylation, precursor maturation, etc.

22

Western Blotting

Lack of controls
While the omission of control samples from a Western blot is nota cause of non-specific bands, their inclusion can tell you why you
may be seeing them on your membrane.
Controls you could include are:•

Positive controls:–– Samples from cells overexpressing the target protein.
–– Purified recombinant protein.
–– Cell line/tissue with proven positive signal.

•

Negative controls:–– Samples targeted with RNA interference.
–– Samples from knockout tissues/cells.
–– Cell line/tissue with proven negative signal.

Other
Blotting Issues

Ghost hollow bands•

This happens when the ECL substrate is used up too rapidly.

Sample overloading•

Decrease the total protein loading for each sample.

Too much antibody•

Decrease the concentrations of the primary and/or
secondary antibodies.

Inverse staining (i.e., white bands on a dark blot)•

Too much primary and/or too much secondary antibody.

•Use antibodies at higher dilutions.

Molecular weight marker staining•

The antibody reacts with the MW marker.

•

Add a blank lane between the MW marker and the firstsample lane.

“Smiling” bands•

Migration through the gel was too hot or too fast.

•

Reduce the voltage applied to run the SDS-PAGE gel or run
the gel in a cold room.

ptglab.com

23

Uneven bands•

High protein concentrations can result in diffuse
protein bands.

•

Uneven protein loading: assay protein samples and load byprotein amount. Check for even protein loading by stripping
and reprobing the blot with an internal control antibody (oruse an HRP-conjugated loading control antibody).

•

Uneven gel composition (gel has set too quickly while
casting or buffer was mixed inadequately).

•

Uneven bands can be due to insufficient buffer being added
to the tank during running.

Blank areas/white spots•

Can be caused by improper/uneven transfer or air bubbles.

Dark spots/dots•

This problem can be caused by antibodies binding to
the blocking reagent in the blocking buffer.

•

Change to another blocking reagent.

•

Filter the blocking buffer.

•

Wash excess detection reagent from the membrane
before exposure.

FROM OUR BENCH  TO YOUR BENCH™
Proteintech products now featurein over 20,000 publications worldwide.
www.ptglab.com

24

Western Blotting

PROMOTION£149 CONTROLANTIBODIESFor a limited time only, Proteintech
control antibodies are just £149 each.*
Order online at ptglab.com*Priced at €149 in Europe.

ptglab.com

25

Antibodies Included:
Antibody Name

Catalog Number

Application

6*His, His Tag

76

66005-1-Ig

ELISA, IF, IP, WB

alpha Tubulin

95

66031-1-Ig

ELISA, FC, IF, IHC, IP, WB

beta Actin

771

60008-1-Ig

ELISA, FC, IF, IHC, WB

beta Tubulin

4210094-1-AP

ELISA, FC, IF, IHC, IP, WB

Flag Tag

15

66008-2-Ig

ELISA, IF, IP, WB

GAPDH

669

60004-1-Ig

ELISA, FC, IF, IHC, IP, WB

66001-1-Ig

ELISA, IP, WB

GST-TAG

The above is a small selection of the antibodies available at the discounted price.
A full list of over 50 antibodies included can be found at ptglab.com.

00

This number shows the amount of times our antibody has been cited in a publication.

26

Western Blotting

Loading Control

ANTIBODIES
Western blotting requires loading controls, they areused for the semi-quantification of protein levels to ensurethat the observed alterations in target proteins are actually
due to experimental manipulations. Internal control proteins,
i.e., those with constant, unchanged levels, are usually detected
in a second round of blotting, following primary detection ofyour protein of interest. This step is used to standardize results
and normalize for any errors that creep into a Western blot
experiment. The loading control candidates for Western blotting
are usually proteins with high, constitutive, and unchanged
expression throughout an experiment, regardless of tissues
or cell types. Control candidates require careful selection as
they can be affected by the conditions of your experiment.
Here, we’ve provided background information on a selectionof the internal control proteins targeted by the control
antibodies we offer.

Actin
Recommended For:
Whole cell/cytoplasma
Molecular Weight~42 kDaThe six isoforms of actin constitute a family of highly conserved
globular proteins comprising three main isoform groups: alpha,beta, and gamma. The alpha actins are a major constituent of thecontractile apparatus in muscle tissues. The beta and gamma
actins coexist in most cell types and are an integral part of thecytoskeleton. Together, actins are the most abundant proteinsin the typical eukaryotic cell, accounting for about 15 percent
of total protein in some cell types. As such, actin is widely used
as an internal control in Western blotting experiments.

Key
Lane 1:

Zebra Fish

Lane 2:

Human Placenta

Lane 3:

Human Heart

50kd

Lane 4:

Human Brain

40kd

Lane 5:

SW1900 Cell Line

Lane 6:

A2780 Cell Line

Lane 7:

PC-3 Cell Line

Lane 8:

HepG2 Cell Line

Lane 9:

HEK293 Cell Line

100kd
70kd

30kd
1

2

3

4

5

6

7

8

9 10 11 12 13 14

Lane 10: HeLa Cell Line
Lane 11: NIH3T3 Cell Line
Lane 12: SP2/0 Cell Line
Lane 13: Raw264.7 Cell Line
Lane 14: C6 Cell Line

Western blot analysis of ACTB in multiple cell lines and tissue lysatesusing anti-beta actin mouse monoclonal (60008-1-Ig) antibody at adilution of 1:5000.

ptglab.com

27

Proteintech’s polyclonal ß-actin antibody (20536-1-AP)
was generated using a ß-actin protein antigen (amino acids
14–167). It recognizes all forms of actin, making it a pan-actin
antibody. However, if your studies involve work with skeletal
muscle samples, or you are working with conditions thatsee changes in cell growth or altered interactions with theextracellular matrix, another loading control may be better
suited to your needs.

Key
100kd
70kd

Lane 1:

1:500

Lane 2:

1:1000Lane 3:

1:2000Lane 4:

1:4000

50kd
40kd

30kd
1

2

3

4

HeLa cell lysate (10 ug/lane) was separated by SDS-PAGE and actin was
detected by anti-ß-actin antibody 20536-1-AP at varying dilutions. (L–R)
1:500, 1:1,000, 1:2,000, and 1:4,000.

Actin Antibodies

Related Antibodies

Cat NoTypeß-actin

Antibody Name
9920536-1-AP

Rabbit Polyß-actin

622

60008-1-Ig

Mouse Mono

Antibody NameCat No

Type

ACTA1

17521-1-AP

Rabbit Poly

55135-1-AP

Rabbit Poly

HRP-60008

Mouse Mono

α-SMA
HRP-conjugatedß-actin00

6This number shows the amount of times our antibody has been cited ina publication.

28

COX-4
Recommended For:
Mitochondrial
Molecular Weight
17 kDaWestern Blotting

COX-4, or COXIV (cytochrome c oxidase subunit IV), is a nuclearencoded subunit of the human mitochondrial respiratory chain
enzyme cytochrome c oxidase (COX). The COX-4 subunit can be
expressed as either of two isoforms. Because of its dependably
high level, COX4I1 is commonly detected as an effective loading
control for mitochondrial proteins. However, some caution isadvised when selecting this protein for Western blot detection
as many other proteins run at its 17 kDa size during SDS-PAGE(make sure your band of interest won’t be obscured). For analternative mitochondrial protein loading control, see ourentry on VDAC1 below.

100kd

70kd
50kd
40kd
30kd
20kd
15kd

10kd

MCF7 cells were subjected to SDS PAGE followed by Western blot with
11242-1-AP(COXIV antibody) at a dilution of 1:1000.

COX-4 Antibodies

Antibody Name
COX4I131

COX4I1
COX4I200

6Cat NoType11242-1-AP

Rabbit Poly

66110-1-Ig

Mouse Mono

11463-1-AP

Rabbit Poly

This number shows the amount of times our antibody has been cited ina publication.

“However, some caution is advised whenselecting this protein for Western blot
detection as many other proteins run atits 17 kDa size during SDS-PAGE.”

ptglab.com

GAPDH
Recommended For:
Whole cell/cytoplasma
Molecular Weight
36 kDa29

Glyceraldehyde 3-phosphate dehydrogenase, commonly
known as GAPDH, catalyzes the six steps of glycolysis. It also
participates in nuclear events. Its expression is high and constantin most tissues and cell types. GAPDH is commonly used as aprotein loading control in Western blotting and internal control
for RT-PCR. However, the amount of GAPDH expression candiffer between some tissues. It is also worth noting that somephysiological factors, such as hypoxia and diabetes, increase
GAPDH expression in certain cell and tissue types.
Proteintech has both monoclonal GAPDH (60004-1-Ig) andpolyclonal GAPDH (10494-1-AP) antibodies available, bothraised against a whole-protein antigen (amino acids 1–335)
of human origin.

Key
100kd
70kd

Lane 1:

1:2000Lane 2:

1:4000Lane 3:

1:5000Lane 4:

1:16000

50kd
40kd

30kd
1

2

3

4

Western blot with HeLa cell lysate using anti-GAPDH (10494-1-AP) at various
dilutions. (L–R) 1:2000, 1:4000, 1:8000, and 1:16000.

GAPDH Antibodies

Related Antibodies

Cat No

Type

GAPDH

Antibody Name
497

10494-1-AP

Rabbit Poly

GAPDH

499

60004-1-Ig

Mouse Mono

Antibody NameCat NoType

HRP-conjugated
GAPDH

HRP-60004

Mouse Mono

00

This number shows the amount of times our antibody has been cited ina publication.

30

Tubulin
Recommended For:
Whole cell/cytoplasma
Molecular Weight
50–55 kDa

Western Blotting

Tubulins are the major components of microtubules. The
Microtubules are involved in a wide variety of cellular activities.
They are highly and stably expressed and conserved across the
species, and tubulins make excellent whole-cell or cytoplasmic
fraction loading controls. However, tubulin expression may vary
according to resistance to antimicrobial and antimitotic drugs.
Proteintech has polyclonal antibodies against several tubulin
subunits, including an α-tubulin antibody (11224-1-AP) andtwo ß-tubulin antibodies, 10068-1-AP and 10094-1-AP.

Key
116kd
66kd

45kd

Lane 1:

HeLa Cell

Lane 2:

NIH3T3 Cell

Lane 3:293 CellLane 4:

A431 Cell

Lane 5:

Jurkat Cell

Lane 6:

Raji Cell

35kd
1

2

3

4

5

6

Western blot on multiple cells with anti-tubulin-alpha (11224-1-AP) at adilution of 1:1,000.

Tubulin Antibodies

Related Antibodies

Cat NoType

Alpha Tubulin

Antibody Name
75

11224-1-AP

Rabbit Poly

Beta Tubulin

1910068-1-AP

Rabbit Poly

Beta Tubulin

34

10094-1-AP

Rabbit PolyCat NoType

3666031-1-Ig

Mouse Mono

HRP-66031

Mouse Mono

Antibody Name
Alpha Tubulin
HRP-conjugated
TUB1A
00

This number shows the amount of times our antibody has been cited ina publication.

ptglab.com

31

CONTACT US
Proteintech GroupUS Head OfficePhone
1 (888) 4PTGLAB(1-888-478-4522)(toll free in USA),or 1(312) 455-8498 (outside USA)
Emailproteintech@ptglab.com

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You can also speak to
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32

Western Blotting