Western Blot Protocol

This step by step protocol provides everything you need to carry out a successful Western Blot. Written by our PhD qualified expert antibody team, this western blot protocol includes advice for planning your western blot, carrying it out safely, analysing your results, as well as recipes for all the solutions you will need, and a troubleshooting guide.

 

Contents

1. Introduction
2. Key Decisions

2.1 Choosing an acrylamide gel percentage
2.1.1 Single concentration gels
2.1.2 Variable concentration gels
2.1.3 Stacking gels

2.2 Choosing a buffer

2.3 Choosing a blocking solution
2.3.1 Milk based blocking solutions
2.3.2 BSA
2.3.3 Fish skin gelatin
2.3.4 Serum

2.4 Selecting appropriate loading controls
2.4.1 What are loading controls? 
2.4.2 Choosing a loading control

 

 

3. Equipment and consumables
3.1 SDS Page
3.2 Electrophoretic Transfer
3.3 Immunoblotting


4. Protocol
4.1 Safety
4.2 SDS-PAGE
4.3 Electrophoretic transfer
4.4 Immunoblotting


5. Analysis
5.1 Measuring the molecular weight of a protein
5.2 Quantifying protein expression from an immunoblot

 

 

 

6.0 Solutions & Recipes
6.1 Buffers

 

 

 

 

6.2 Gel formulations


6.3 Blocking solutions

7. Troubleshooting


8. Further reading

Download pdf Western Blot Protocol

 

1. Introduction

Western blotting, also known as immunoblotting, is a key technique in molecular biology to investigate changes in protein expression in a range of different tissue types. Proteins are first denatured before being loaded onto an acrylamide gel with an electric current applied. Proteins are separated by their mass before being transferred onto a membrane (either PDVF or nitrocellulose) and probed with antibodies to reveal expression of specific proteins.

 

2. Key Decisions

Before starting any western blot experiment it is important to consider the following points:

  • What percentage acrylamide gel to use in order to get the best separation (see section 2.1)
  • What blocking solution to use for the experimental conditions (see section 2.2)
  • What loading controls should be included to account for variability in loading and transfer (see section 2.3)

 

2.1 Choosing an acrylamide gel percentage

2.1.1 Single concentration gels

In a SDS page all proteins are denatured and have a uniform negative charge applied to them therefore migration is due to differences in size. Polyacrylamide gels are a matrix of cross-linked acrylamide monomers with the tightness of the mesh dependent upon the amount of acrylamide and cross-linker present. Different sized proteins therefore require different formulations of acrylamide gel to get optimum separation (figure 1).

Protein size (kDa)

Gel Percentage (%)

4-40

20

12-45

15

10-70

12

15-100

10

25-200

7.5

>200

5


Table 1 Suggested concentrations of acrylamide gel depending upon protein of interest size.
Where multiple proteins of differing sizes need to be separated consider using a gradient gel.

 

Where there is only one protein of interest or the proteins to be separated are of a similar size then a single concentration gel can be used. Generally the larger the protein the larger pore size is needed in the polyacrylamide gel and the smaller the protein the smaller the pore size (table 1). Once the gel concentration needed has been identified these can either be purchased as pre-cast gels or made in the laboratory using the recipe in table 2.

 

Reagent

Order

Gel concentration (%)

20

15

12

10

7.5

5

dH2O

1

0.93 ml

2.34 ml

3.28 ml

3.98 ml

4.78 ml

5.61 ml

1.5M Tris-HCl pH 8.8

2

2.5 ml

2.5 ml

2.5 ml

2.5 ml

2.5 ml

2.5 ml

10% SDS

3

100 µl

100 µl

100 µl

100 µl

100 µl

100 µl

30% Acrylamide/Bis (29.2:0.8)

4

6.7 ml

5 ml

4 ml

3.3 ml

2.5 ml

1.67 ml

10% APS

5

50 µl

50 µl

50 µl

50 µl

50 µl

50 µl

TEMED

6

10µl

10µl

10µl

10µl

10µl

10µl


Table 2 Recipe for the construction of polyacrylamide resolving gels.
Makes a 10ml gel. Be sure to add reagents in the correct order with APS and TEMED being added last. CAUTION: Acrylamide is a potent neurotoxin therefore gloves should be worn at all times.
 
 

2.1.2 Variable concentration gels

If multiple proteins of significantly differing sizes need to be separated then gradient gels can be instead used. These vary the concentration of the gel along the migration path to provide optimal separation. These can be made in the laboratory but are more easily purchased.

 

2.1.3 Stacking gels

In order to line up proteins before they enter the resolving gel it is important for proteins to pass through a short layer of stacking gel (Figure 1). This allows proteins to enter the resolving gel at the same point and is the same acrylamide concentration regardless of protein size (Table 3).

Structure of an acrylamide gel for SDS-PAGE

Figure 1. Structure of an acrylamide gel for SDS-PAGE

 

Reagent

Order

Volume

dH2O

1

3.05 ml

0.5M Tris-HCl pH 6.8

2

1.25 ml

10% SDS

3

50µl

30% Acrylamide/Bis (29.2:0.8)

4

650µl

10% APS

5

25µl

TEMED

6

10µl


Table 3 Recipe for acrylamide stacking gel.
Makes 5ml suitable for a 10ml resolving gel. Be sure to add reagents in the correct order with APS and TEMED being added last. CAUTION: Acrylamide is a potent neurotoxin therefore gloves should be worn at all times when making or handling the gel.

 

2.2 Choosing a buffer

Western blots are carried out using either phosphate buffered saline (PBS) or Trizma buffered saline (TBS). While these can often be used interchangeably it is important to consider key differences between the different buffers and make a choice of buffer accordingly (Table 4).

Factor

PBS

TBS

Temperature pH stability

Relatively stable

pH can be highly temperature dependent

Suitability for use with phosphorylation specific antibodies

Not suitable

Suitable

Suitable for use with alkaline phosphatase labelled antibodies

Not suitable

Suitable

Cost

Lower

Higher

Suitable for use with living cells

Suitable

Not suitable


Table 4 Comparison between PBS and TBS buffers.

 

2.3 Choosing a blocking solution

It is important to choose the correct blocking solution dependent on what experiment is being carried out and what antibodies are being used. For example BSA and milk contain phosphotyrosine which will interrupt experiments using phosphorylation specific antibodies. Additionally alkaline phosphatase can be inhibited by certain milk preparations. For a summary of the different blocking solutions see table 5.

Blocking solution

Constituents

Advantages

Disadvantages

Milk based

5% non-fat dry milk in PBS-T / TBS-T

  • Cheap
  • Most popular solution used by researchers
  • Clean background
  • Not suitable for phosphorylation specific antibodies
  • Not suitable for biotin labelled antibodies
  • Can interfere with alkaline phosphatase labelled antibodies
  • Needs making up fresh and degrades rapidly

BSA

3% BSA in PBS-T / TBS-T

  • Good signal strength
  • Suitable for all antibodies
  • Relatively expensive

Fish skin gelatin

1% fish skin gelatin in PBS-T / TBS-T

  • Cheap
  • Doesn’t contain mammalian proteins therefore low background
  • Cannot be used with biotin labelled antibodies

Serum

10% serum in PBS-T / TBS-T

  •  Clean background
  • Expensive
  • Incompatible with some anti-immunoglobulin antibody detection.
  • Contains immunoglobulins and serum proteins that can cross-react with the primary or secondary antibody


Table 5. Comparison between commonly used blocking buffers in western blotting.

 

2.2.1 Milk based blocking solutions

The most commonly used blocking solution is a solution of 5% non-fat dry milk in PBS-T which works well for the vast majority of applications at a inexpensive price. Some preparations contain sodium azide as a preservative although it should be noted that azide can inhibit horseradish peroxidase, the most commonly used secondary antibody conjugate in western blots. Due to the rapid growth of microbial contamination milk based solutions should be made up fresh for every application. Milk based blocking solutions should not be used for any experiments involving phosphorylation specific antibodies due to the presence of casein which is a phosphoprotein that can interfere with detection. Alkaline phosphatase antibodies can also be inhibited by some preparations of milk alongside biotin labelled antibodies being interfered with by the milk preparation.

 

2.3.2 BSA

BSA is also widely used as it is suitable for all detection systems as it does not contain biotin or phosphopeptides. There can however be issues with contamination from native immunoglobulins which can cause issues with cross-reactivity. BSA is normally prepared as a 3% dilution in PBS-T or TBS-T. The main drawback of BSA usage is it’s relatively high cost compared to other blocking solutions.

 

2.3.3 Fish skin gelatin

Fish skin gelatin based blocking solutions offer an excellent alternative to other blocking solutions due to its lack of mammalian proteins which reduces the risk of antibody cross-reactivity with the blocking buffer. Fish skin gelatin is also cheap however it should be noted that it isn’t appropriate for use with biotin labelled antibodies due to it containing biotin. Fish skin gelatin is normally used as a 1% solution in PBS-T or TBS-T and has the advantage of remaining liquid at room temperature compared to porcine gelatin.

 

2.3.4 Serum

Serum is another option when considering which blocking solution to use however is declining in popularity due to its high cost and the risk of cross-reactivity with immunoglobulins present in the serum. It has however been reported to offer a clean background and is commonly used as a 10% dilution in either PBS-T or TBS-T.

 

2.4 Selection of appropriate loading controls

2.4.1 What are loading controls?

  • Loading controls are antibodies against a different target to the protein of interest used in immunoblotting (Western blotting). These targets are often highly expressed housekeeping proteins who’s expression is stable. Loading controls are essential when the relative expression of proteins is being compared in a gel and are used to:
  • Ensure that the loading of proteins is uniform across the whole gel. Where there has been unequal loading the loading control can be used to account for this.
  • Ensure that there has been equal transfer of proteins from gel to membrane across the gel

 

2.4.2 Choosing a loading control

There are several key principles that need to be followed when choosing a loading control for immunoblotting:

  • The loading control and target of interest should have different molecular weights to ensure they do not overlap on the gel
  • The loading control expression should not be effected by any experimental manipulation between samples.
  • The loading control should have high levels of expression in the sample
  • The loading control should be in the portion of the gel where there is linear separation otherwise it will not be possible to quantify it.

Use table 6 to help identify a suitable loading control depending on molecular weight of the target protein and the subcellular location of the sample. You can view all the loading controls available from Hello Bio here.

 

Molecular weight (kDa)

Whole cell

Mitochondrial

Nuclear

Membrane

Cytoskeleton

Serum

125

Vinculin

 

 

 

 

 

110

 

 

 

NaK ATPase

 

 

75

 

 

 

 

 

Transferrin

66

 

 

Lamin B1

 

 

 

60

 

HSP60

HDAC1

 

 

 

55

 

 

 

 

 

 

50

Alpha tubulin

 

 

 

Alpha tubulin

 

Beta tubulin

 

 

 

Beta tubulin

 

45

Actin

 

YY1

 

Actin

 

40

Beta actin

 

 

 

Beta actin

 

35

GAPDH

 

TBP

 

 

 

30

 

VDAC1/Porin

PCNA

 

 

 

20

 

Cyclophilin B

 

 

 

 

 

Cofilin

COX IV

 

Cofilin

 

15

 

 

Histone H3

 

 

 


Table 6. Common loading controls.
Molecular weight is of the loading control therefore choose one at a different mass to the target of interest.

 

3. Equipment and consumables

Assuming a base level of standard laboratory equipment (e.g. pipettes, de-ionised water system, measuring cylinders etc) the specific equipment and consumables you will require for western blotting are detailed by stage within this section.

3.1 SDS Page

Equipment

Consumables

Powerpack

Protein ladder

Gel tank and cassette

Filter papers

Casting plates

Running buffer

Combs (same thickness as casting plates)

Sample buffer

Casting stand and gel holders

Ethanol

Heating block (able to achieve 85ºC)

Resolving gel

Microcentrifuge

Stacking gel

Loading tips (not essential but make gel loading easier)

 

 

3.1 Electrophoretic transfer

Equipment

Consumables

Powerpack

Transfer buffer

Gel tank and cassettes

Filter papers

Large shallow tray (for preparing transfer cassettes in)

PVDF membrane

Roller (for removing bubbles in transfer sandwich with)

Methanol

Absorbent pads

 

Ice pack

 

Magnetic stirring plate and stirring bars

 

Gel opening tool

 

 

3.3 Immunoblotting

Equipment

Consumables

Rocker

PBS-T

Containers for putting membranes in

Blocking solution

Heat sealer

Primary antibody

Detection system (e.g. film with darkroom or gel imaging system)

Secondary antibody

 

Plastic bags for heat sealer

 

Enhanced chemiluminescence substrate (ECL substrate)

 

Optional: Ponceau stain

 

 

4. Protocol

4.1 Safety

Many of the chemicals used in western blotting have dangerous properties and can cause serious harm if not handled correctly. Always follow local rules and read the full COSHH document for any chemical that you have not used previously. Always wear appropriate PPE such as a lab coat and gloves.

Specifically highlighted hazards:

  • Acrylamide is an extremely toxic neurotoxin and carcinogen in monomeric forms. Wipe down benches after use and dispose of gels and any contaminated waste into separated acrylamide waste.
  • Beta-mercaptoethanol is toxic and needs disposing separately to normal laboratory waste.

 

4.2 SDS Page

1. Clean casting stand and gel holders with distilled water. Clean plates with distilled water.

2. Construct gel mould on holder

    1. Place the casting frame upright with the pressure cams in the open position and facing forward on a flat surface.
    2. Select a spacer plate of the desired gel thickness and place a short plate on top of it.
    3. Orient the spacer plate so that the labelling is up. Slide the two glass plates into the casting frame, keeping the short plate facing the front of the frame (side with pressure cams)
    4. When the glass plates are in place, engage the pressure cams to secure the glass cassette sandwich in the casting frame. Check that both plates are flush at the bottom.
    5. Place the casting frame into the casting stand by positioning the casting frame (with the locked pressure cams facing out) onto the casting gasket while engaging the spring-loaded lever of the casting stand onto the spacer plate.
  1. It is often a good idea to fill the gel plates with dH2O to check for leaks while making the resolving gel. Pour out when ready to pour the resolving gel.

3. Mix resolving gel (adding ammonium persulphate and TEMED last) and pour immediately. The resolving gel should be far enough below the top of the gel plates to allow insertion of the comb + 1cm.

Reagent

Order

 

Gel concentration (%)

20

15

12

10

7.5

5

dH2O

1

0.93ml

2.60ml

3.59ml

4.26ml

5.09ml

5.93ml

1.5M Tris-HCl pH 8.8

2

2.5ml

2.5ml

2.5ml

2.5ml

2.5ml

2.5ml

10% SDS

3

100µl

100 µl

100 µl

100 µl

100 µl

100 µl

30% Acrylamide/Bis (29.2:0.8)

4

6.66ml

5.00ml

4.00ml

3.33ml

2.50ml

1.67ml

10% APS

5

50µl

50µl

50µl

50µl

50µl

50µl

TEMED

6

10µl

10µl

10µl

10µl

10µl

10µl


Optional: Degas the mixture using a tap vacuum setup for 10 minutes (make sure to remove bung before turning off tap)

4. Overlay resolving gel gently with water or ethanol to maintain a flat surface on the resolving gel during polymerisation. Leave to set for 15/20 minutes.

Tip: Make excess gel and then monitor the polymerisation using this.

5. Mix stacking gel except APS and TEMED. Pour off overlay and use a tissue wick to remove the last remnants. Add APS and TEMED to stacking gel and pour. Add the comb with no air bubbles and leave to set for around 20 minutes.

Reagent

Order

Volume

1 gel

2 gels

3 gels

4 gels

5 gels

dH2O

1

3.05ml

6.10ml

9.15ml

12.2ml

15.25ml

0.5M Tris-HCl pH 6.8

2

1.25ml

2.5ml

3.75ml

5ml

6.25ml

10% SDS

3

50µl

100 µl

150 µl

200 µl

250 µl

30% Acrylamide/Bis (29.2:0.8)

4

650µl

1.3ml

1.95ml

2.6ml

3.25ml

10% APS

5

25µl

50µl

75µl

100µl

125µl

TEMED

6

10µl

20µl

30µl

40µl

50µl

Optional: Degas the mixture using a tap vacuum setup for 10 minutes (make sure to remove bung before turning off tap)

6. Heat samples in sample buffer for 10 minutes at 85⁰C. Give samples a quick spin before loading to remove debris.

7. Make up 1l of 1X running buffer (diluting 10X buffer as necessary). Construct running assembly making sure seals are tight (use grease if necessary). Add running buffer to top tank (above level of gel, CATHODE). Check for leaks. Add running buffer to bottom tank (ANODE)

8. Remove comb. Gently wash out each well

9. Add samples in sample buffer. Ideally do not use outside wells. Run markers in central well and at side. Any unused wells should be filled with a small volume of 1X sample buffer. Sample loading volumes should be from 5 μL–35 μL per lane (depending on gel).

  1. Make sure a consistent quantity of protein is loaded per well ≈ 20µg.
  2. Use around 4-5µl of marker, one option is to load 2µl of marker on one side of the gel and 5µl on the other to distinguish sides.
  3. Mix marker with sample buffer to make up to the same loading volume as samples

10. Connect up equipment (red to red, black to black)

11. Run at 60V until dye front is in the resolving gel (30 minutes) and then run at 120-150V in gel (45-90 minutes). Vary by experience and do not exceed 200V.

12. Stop when gel front nears bottom.

 

4.3 Electrophoretic transfer

1. Cut out a rectangle of PDVF membrane using a template to fit the size of the gel. Write the date and name of experiment on the membrane in pen

2. Wash membrane with methanol for 30 seconds (methanol can be reused)

3. Wash membrane with transfer buffer

4. Soak the blotting paper and sponges in transfer buffer

5. Put cassette (red and black) into tank

6. Take gel out of running tank and carefully open the plates; cut off the stacking gel and put in acrylamide waste

7. Soak the gel in transfer buffer

8. Assemble the cassette in the following order making sure to keep everything as wet as possible:

  1. Black side of cassette
  2. Sponge
  3. 3x blotting paper
  4. Gel face down (so the ladder is on the right side of the gel with the red marker nearer the top)
  5. Membrane also face down
  6. 3x blotting paper (after this step you can use a roller to gently get rid of any bubbles between the gel and the membrane)
  7. Sponge
  8. Clear side of cassette

9. Close the cassette and put into the holder making sure the colours align. The proteins will run towards the positive anode (red)

10. Add the ice block and stirring bar

11. Fill up the tank with transfer buffer without it overflowing

12. Turn on stirring plate and put on lid

13. Set the power pack to 400 mA for 90 minutes (check there are bubbles before leaving). Pause point: The membranes can be left in transfer buffer overnight for processing the next day however this means that the entire protocol will take 3 days as opposed to 2 if the primary antibody addition is achieved on day 1.

4.4 Immunoblotting

1. Rinse the membrane several times with PBS-T.

2. Add a blocking solution. The choice of blocking will depend upon the target and upon the detection method. E.g. alkaline phosphatase is inhibited by some milk preparations.

3. Incubate with agitation for 2hrs

4. Add the primary antibody solution. All antibodies should be diluted in blocking solution. Incubate overnight with agitation in the cold room.

  1. Add the antibody to strip to a plastic bag then use a heat sealer to seal the pouch. Doing this should mean only around 1ml per bag is needed.

5. Wash the blot with 3 quick changes of PBS-T then 3 times for 5 minutes under agitation.

6. Add the secondary antibody to blocking solution. Incubate for 1-2hrs at room temperature with agitation. For fluorescently labelled secondary antibodies all steps should be done in the dark from now on.

7. Wash the blot with 3 quick changes of PBS-T then 3 times for 5 minutes under agitation.

8. Detect using ECL (for HRP conjugated secondaries). Follow manufacturer instructions and cover membrane with transparent plastic sheet to stop it drying out.

  1. Detection can be either through the use of film and a darkroom or by using a gel imaging system.
  1. Film: Generally the most sensitive but can be expensive and it is more difficult to use for quantitative measurements
  2. Imaging system: Once initial outlay has been paid is much cheaper and allows accurate quantitative measures. However can be less sensitive than film.

 

4.5 Stripping and Re-probing

Following the completion of an immunoblot it is possible to remove the primary and secondary antibodies then re-probe the membrane for a new target. This is commonly used with a loading control antibody following probing for a protein of interest. The same membrane can be stripped and re-probed multiple times, however each treatment removes protein from the membrane which should be taken into account. This protocol requires a PVDF membrane and detection using ECL.

1. Incubate membrane in two changes of stripping buffer at room temperature for 10 minutes an incubation.

2. Wash membrane in 3 quick changes of PBST

3. Proceed to blocking (step 2 of section 4.4)

If you want to check that stripping has been successful then incubate membrane with ECL detection substrates after step 2 then image. The bands from the previous antibody should have disappeared and there be a clear uniform background.

 

5. Analysis

5.1 Measuring the molecular weight of a protein

The molecular weight of a protein can be estimated by comparing the migration of proteins of known molecular weight (such as in a protein ladder) and the target protein. A general procedure for doing this is:

      1. Run the gel using a molecular weight ladder, transfer to the membrane and then visualise the proteins using a dye such as Coomassie blue or Ponceau.
      2. Calculate the relative migration distance (Rf) of each protein standard and the target protein using the equation below. This can either be measured using a ruler or within appropriate software.

        Formula

    1. Plot the log(MW) of the protein standards against relative migration distance (Rf) on a graph and generate a curve of best fit. This should be linear if the samples are fully denatured and the gel percentage was adequate for sufficient separation.
    2. Use the equation of the best fit line to calculate the mass of the target using its Rf. The general equation is: general equationwhich in figure 2 simplifies to: simplified equation
    3. The actual protein molecular weight as determined by mass spectrometry is likely to differ from the estimated through western blotting due to differences in glycosylation status, experimental inaccuracies and some proteins not being amenable to full denaturation by SDS.

Standard curve of log MW against relative migration Rf
Figure 2. Example standard curve of log(MW) against relative migration (Rf). A high R2 value indicates increased accuracy in interpretation of the unknown proteins MW.

 

5.2 Quantifying protein expression from an immunoblot

Protein expression can be quantitatively compared within a immunoblot using densitometry. Crucially this technique can only inform of relative changes in abundance between samples therefore without known standards, cannot be used to give exact concentrations.

 

  • Before trying to take any quantitative measurements it is critical to make sure that the signal is not saturated and is in its linear range (figure 3). If the signal is saturated then it isn’t possible to accurately compare different protein abundances. However, saturation is a relatively easy problem to solve as by reducing the exposure time this can be avoided. However, where there are large differences in protein abundance between samples then it may be necessary to re-attempt the western blot with more similar protein concentrations if it isn’t possible to have both bands properly exposed (i.e. one is always saturated to see the other). Reducing the concentration of primary antibody is also another way of reducing saturation.

 

Graph to show the saturation of western blot signal at high levels of bound protein

Figure 3: Example curve showing the saturation of western blot signal at high levels of bound protein. Above each datapoint is a representative illustration of the band seen. Adapted from Bell, 2016. BMC Biol; 14:116.

 

  • It is next important to subtract any background noise from the image. Differences in background across the blot may not be consistent leading to changes in band darkness that are influenced by background, not signal intensity. This step is easily achieved in both bespoke analysis software and freeware such as ImageJ (https://imagej.nih.gov/).
  • Next it is critical to normalise the signal intensity for protein loading. Naturally each lane will have a slightly different amount of protein in it therefore by normalising to expression of stably expressed “housekeeping” proteins such as GAPDH or β-actin this can help to ensure that any changes in expression of a target protein are not just due to differences in loading. It is important when designing the experiment to think about whether the manipulation might affect expression of the loading control and another issue is that due to high expression it is often difficult to capture them in their linear range.
  • Finally it is important to subject results to the appropriate statistical tests for the experimental design. It should be planned before the experiment was carried out what n-number was needed to achieve a sufficient statistical power (often chosen as 0.8).

 

6. Solutions & Recipes

6.1 Buffers

0.5M Tris-HCl pH 6.8

Note: For longer term storage autoclave then store at 4ºC

Reagent

Amount to add

100ml

500ml

1000ml

Tris-base

6.06g

30.29g

60.57g

dH2O

≈80ml

≈400ml

≈800ml

Conc. HCl

Adjust pH to 6.8

dH2O

Make up to final volume required

 

 

1.5M Tris-HCl pH 8.8

Note: For longer term storage autoclave then store at 4ºC

 

Reagent

Amount to add

100ml

500ml

1000ml

Tris-base

18.17g

90.86g

181.71g

dH2O

≈80ml

≈400ml

≈800ml

Conc. HCl

Adjust pH to 8.8

dH2O

Make up to final volume required

 

 

10x running buffer

Notes:
  1. For 1x buffer dilute with dH­­­2O in a 1:10 dilution
  2. SDS powder is a potent respiratory irritant therefore should be weighed out in the fume hood or with breathing protection.
  3. Store at room temperature

 

Reagent

Amount to add

Final concentration

500ml

1000ml

2000ml

Glycine

72g

144g

288g

1.91M

Tris-base

15g

30g

60g

247mM

SDS

5g

10g

20g

35mM

dH2O

Make up to final volume required

 

 

 

10x transfer buffer

Notes:
  1. Store at room temperature
  2. For 1x buffer dilute 1:10 with dH2O / 20% methanol 

Reagent

Amount to add

Final concentration

500ml

1000ml

2000ml

Glycine

72g

144g

288g

1.91M

Tris-base

15g

30g

60g

247mM

dH2O

Make up to final volume required

 

 

10% SDS

Notes:
  1. Store at room temperature
  2. Over time SDS may precipitate out of solution. If this occurs remix until all SDS has re-dissolved.

Reagent

Amount to add

100ml

200ml

500ml

SDS

10g

20g

50g

dH2O

Make up to final volume required

 

 

2x Sample loading buffer

Note: store in aliquots at -20ºC
 

Reagent

Amount to add

Final concentration

5ml

50ml

100ml

10% SDS

2ml

20ml

40ml

4%

0.2% Bromophenol blue

0.1ml

1ml

2ml

0.004%

Glycerol

1ml

10ml

20ml

20%

0.5M Tris-HCl pH 6.8

1.25ml

12.5ml

25ml

0.125M

ß-mercaptoethanol

0.5ml

5ml

10ml

10%

H2O

0.15ml

1.5ml

3ml

-

 

 

10% APS

Note: store in aliquots at -20ºC
 

Reagent

Amount to add

10ml

50ml

100ml

Ammonium persulphate

1g

5g

10g

dH2O

Make up to final volume required

 

 

 

10x PBS

Note: store at room temperature
 

Reagent

Amount to add

Final concentration

500ml

1000ml

2000ml

NaCl

40g

80g

160g

1.37M

KCl

1g

2g

4g

27mM

Na2HPO4

7.2g

14.4g

28.8g

100mM

KH2PO4

1.2g

2.4g

4.8g

20mM

dH2O

≈400ml

≈800ml

≈1600ml

-

Conc HCl

Adjust to pH 7.4

-

dH2O

Make up to final volume required

-

 

10x TBS

Note: store at room temperature
 

Reagent

Amount to add

Final concentration

500ml

1000ml

2000ml

NaCl

40g

80g

160g

1.37M

Tris-base

12.1g

24.2g

48.5g

200mM

dH2O

≈400ml

≈800ml

≈1600ml

-

Conc HCl

Adjust to pH 7.4

-

dH2O

Make up to final volume required

-

 

 

1x PBST / TBST

Notes:

  1. Tween20 is extremely viscous therefore it is often helpful to cut the end off the pipette tip using scissors to made pipetting easier.
  2. The solution will need a good mixing with a stirring bar before being ready to use
  3. Generally make up fresh and don’t keep for longer than a few days.

Reagent

Amount to add

Final concentration

500ml

1000ml

2000ml

10x TBS / PBS

50ml

100ml

200ml

1x

dH2O

450ml

900ml

1800ml

-

Tween20

0.5ml

1ml

2ml

0.1%

 

Stripping buffer

Notes: 

  1. The buffer can sometimes go cloudy over time however this does not appear to impact it’s effectiveness. 
  2. Store at room temperature
  3. Tween20 is extremely viscous therefore it is often helpful to cut the end off the pipette tip using scissors to made pipetting easier.
Reagent Amount to add Final concentration
100ml 500ml 1000ml
Glycine 1.5g 7.5g 15g 200mM
SDS 0.1g 0.5g 1g 0.1%
Tween20 1ml 5ml 10ml 1%
dH2O ≈80ml ≈400ml ≈800ml -
Conc HCl Adjust to pH 2.2 -
dH2O Make up to final volume required -


Gel formulations

Note: Make sure to add reagents in the order indicated. Acrylamide is a potent neurotoxin therefore take appropriate precautious and wear suitable PPE.

 

5% resolving gel

For proteins larger than 200kDa.
 
Reagent Order Gels Unit
1 2 3 4 5 6 7 8
dH2O 1 5.93 11.85 17.78 23.70 29.63 35.55 41.48 47.40 ml
1.5M Tris-HCl
pH 8.8
2 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 ml
10% SDS 3 100 200 300 400 500 600 700 800 µl
30% Acrylamide/Bis (29.2:0.8) 4 1.67 3.33 5.00 6.66 8.33 9.99 11.66 13.32 ml
10% APS 5 50 100 150 200 250 300 350 400 µl
TEMED 6 10 20 30 40 50 60 70 80 µl

 

7.5% resolving gel

For proteins sized between 25 and 200 kDa.
 
Reagent Order Gels Unit
1 2 3 4 5 6 7 8
dH2O 1 5.09 10.19 15.28 20.37 25.46 30.56 35.65 40.74 ml
1.5M Tris-HCl
pH 8.8
2 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 ml
10% SDS 3 100 200 300 400 500 600 700 800 µl
30% Acrylamide/Bis (29.2:0.8) 4 2.50 5.00 7.49 9.99 12.49 14.99 17.48 19.98 ml
10% APS 5 50 100 150 200 250 300 350 400 µl
TEMED 6 10 20 30 40 50 60 70 80 µl

 

10% resolving gel

For proteins sized between 15 and 100kDa.
 
Reagent Order Gels Unit
1 2 3 4 5 6 7 8
dH2O 1 4.26 8.52 12.78 17.04 21.30 25.56 29.82 34.08 ml
1.5M Tris-HCl
pH 8.8
2 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 ml
10% SDS 3 100 200 300 400 500 600 700 800 µl
30% Acrylamide/Bis (29.2:0.8) 4 3.33 6.66 9.99 13.32 16.65 19.98 23.31 26.64 ml
10% APS 5 50 100 150 200 250 300 350 400 µl
TEMED 6 10 20 30 40 50 60 70 80 µl

 

12% resolving gel

For proteins sized between 10 and 70kDa.
 
Reagent   Order Gels Unit
1 2 3 4 5 6 7 8
dH2O   1 3.59 7.19 10.78 14.38 17.97 21.56 25.16 28.75 ml
1.5M Tris-HCl
pH 8.8
  2 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 ml
10% SDS   3 100 200 300 400 500 600 700 800 µl
30% Acrylamide/Bis (29.2:0.8)   4 4.00 7.99 11.99 15.98 19.98 23.98 27.97 31.97 ml
10% APS   5 50 100 150 200 250 300 350 400 µl
TEMED   6 10 20 30 40 50 60 70 80 µl

 

15% resolving gel

For proteins sized between 12 and 45kDa.
 
Reagent Order Gels Unit
1 2 3 4 5 6 7 8
dH2O 1 2.60 5.19 7.79 10.38 12.98 15.57 18.17 20.76 ml
1.5M Tris-HCl
pH 8.8
2 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 ml
10% SDS 3 100 200 300 400 500 600 700 800 µl
30% Acrylamide/Bis (29.2:0.8) 4 5.00 9.99 14.99 19.98 24.98 29.97 34.97 39.96 ml
10% APS 5 50 100 150 200 250 300 350 400 µl
TEMED 6 10 20 30 40 50 60 70 80 µl

 

20% resolving gel

For proteins between 4 and 40kDa.
 
Reagent Order Gels Unit
1 2 3 4 5 6 7 8
dH2O 1 0.93 1.86 2.79 3.72 4.65 5.58 6.51 7.44 ml
1.5M Tris-HCl
pH 8.8
2 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 ml
10% SDS 3 100 200 300 400 500 600 700 800 µl
30% Acrylamide/Bis (29.2:0.8) 4 6.66 13.32 19.98 26.64 33.30 39.96 46.62 53.28 ml
10% APS 5 50 100 150 200 250 300 350 400 µl
TEMED 6 10 20 30 40 50 60 70 80 µl

 

Stacking gel

Note: Make sure to add in the order indicated. Acrylamide is a potent neurotoxin therefore take appropriate precautious and wear suitable PPE.

Reagent

Order

Gels

Unit

1

2

3

4

5

6

7

8

dH2O

1

3.05

6.10

9.15

12.2

15.25

18.3

21.35

24.4

ml

0.5M Tris-HCl pH 6.8

2

1.25

2.5

3.75

5

6.25

7.5

8.75

10

ml

10% SDS

3

50

100

150

200

250

300

350

400

µl

30% Acrylamide/Bis (29.2:0.8)

4

650

1.3

1.95

2.6

3.25

3.9

4.55

5.2

ml

10% APS

5

25

50

75

100

125

150

175

200

µl

TEMED

6

10

20

30

40

50

60

70

80

µl

 

6.3 Blocking solutions

See section 2.2 for advice on choosing a blocking solution.
 

Blotto

Note:
  1. Make up fresh for every application
  2. For longer term incubations it is possible to add sodium azide to a final concentration of 0.01% to prevent bacterial growth.

Reagent

Amount to add

Final concentration

10ml

20ml

50ml

1x PBST/TBST

10ml

20ml

50ml

-

Non-fat dry milk (NFDM)

0.5g

1g

2.5g

5%

 

BSA

Note:

  1. Make up fresh for every application
  2. For longer term incubations it is possible to add sodium azide to a final concentration of 0.01% to prevent bacterial growth.

Reagent

Amount to add

Final concentration

10ml

20ml

50ml

1x PBST/TBST

10ml

20ml

50ml

-

Bovine serum albumin (BSA)

0.3g

0.6g

1.5g

3%

 

Fish skin gelatin

Note:

  1. Make up fresh for every application
  2. For longer term incubations it is possible to add sodium azide to a final concentration of 0.01% to prevent bacterial growth.
Reagent Amount to add Final concentration
10ml 20ml 50ml
1x PBST/TBST 10ml 20ml 50ml -
Fish skin gelatin 1g 2g 5g 1%

 

Serum

Note:

  1. Make up fresh for every application
  2. For longer term incubations it is possible to add sodium azide to a final concentration of 0.01% to prevent bacterial growth.
Reagent Amount to add Final concentration
10ml 20ml 50ml
1x PBST 9ml 18ml 45ml -
Serum from same species as secondary antibody 1ml 2ml 5ml 10%

 

7. Troubleshooting

Western blotting is a long multi-step process with many different factors that need to be considered. At some point it is inevitable that something will go wrong or not be optimal. Below are compiled some of the most common pitfalls that can cause western blots to not work.

Problem

Potential cause

Suggested solutions

No bands or faint signal

Incomplete transfer of proteins from the acrylamide gel to PVDF membrane

  • Use a reversible stain such as Ponceau to check that transfer was successful. 
  • Alternatively use Coomassie stain on the gel following transfer to make sure no to little protein remains.
  • Increase the transfer time if short
  • Check that the transfer sandwich was assembled in the correct order so that proteins weren’t transferred away from the membrane 

Over-transfer of proteins through PVDF membrane if transfer is for too long with a low molecular weight target 

  • Reduce either the transfer time or transfer current
  • Choose a PVDF membrane optimised to small proteins

Reagents may have deteriorated due to meeting use by dates or improper storage conditions

  • Make sure all reagents are stored following manufacturer suggestions and are within date

Incorrect species of secondary antibody

  • Ensure that the secondary antibody used is specific for the species the primary is raised in

Buffers are contaminated by bacteria

  • Make sure all solutions are free from contamination. An easy way to check is to ensue that there is no cloudiness visible when solutions are swirled.

Exposure time too short

  • Try increasing the exposure time

Antibody concentration is too low

  • Increase the primary antibody concentration and incubate for a longer amount of time

High background

Contaminated blocking buffer

  • Do not re-use blocking buffers, make up fresh each time

Too high antibody concentration

  • Reduce primary and secondary antibody concentrations

Insufficient washing

  • Increase the number and duration of washing steps after the secondary antibody incubation

Too long exposure

  • Reduce exposure

Antibody has lost specificity due to improper storage

  • Use fresh antibody or freshly thawed aliquots that have been stored at -80°C

Buffers are contaminated by bacteria

  • Make sure all solutions are free from contamination. An easy way to check is to ensue that there is no cloudiness visible when solutions are swirled.

Smears on blot

Blot contamination from touching membrane

  • Only handle membrane as little as possible using the edges, use forceps where able.

Uneven incubations

  • Make sure a rocker or shaker is used for all incubations to ensure that the membrane surface is thoroughly covered in solution.

Parts of membrane dried out

  • Ensure that there is always an excess of liquid at all stages in the process.

Black spots on blot

Antibodies binding to clumped blocking reagent or blocking reagent clumped to membrane

  • Make sure blocking buffers are thoroughly vortexed before use
  • Use fresh blocking buffers

Aggregated secondary antibody

  • Centrifuge secondary antibody to remove aggregates before use.

Acrylamide gel stuck to membrane

  • Ensure all gel contamination is removed after transfer before further processing.

Smile shaped bands

Electrophoresis voltage was too high

  • Reduce voltage
  • Run gel using chilled buffers, in cold room or using an ice pack

Unevenly run bands

Poor acrylamide polymerisation

  • Ensure that gel is fully polymerised following recommended recipe before sample loading.

Blurry bands

Electrophoresis voltage was too high

  • Reduce voltage
  • Start at a lower voltage then turn up after 30 minutes

White centred bands (ECL detection)

Rate of reaction was too high and ECL has been depleted

  • Reduce primary antibody concentration
  • Reduce exposure
  • Reduce secondary antibody concentration

 Extra bands

Target protein has multiple splice variants or isoforms

  • Check in literature for previously reported splice variants.
  • See if a splice variant specific antibody is available.

Target protein has been subject to proteolytic cleavage

  • Try including protease inhibitors to the lysis buffer when preparing samples.
  • Make sure samples are prepared on ice
  • Try using a fresh sample

Antibody epitope is present in another protein

  • Ensure a negative control sample has been included.
  • Try a different antibody using a different epitope
  • Decide if the extra band is an issue for the planned experiment.

Secondary antibody concentration too high

  • Reduce secondary antibody concentration
  • Try a secondary only blot to check non-specific binding of the secondary

Native IgGs present within samples detected by secondary

  • Use pairs of antibodies with no relation to the species the samples come from
  • Decide if this is an impediment to the designed experiment 

Contaminant IgGs within primary antibody reacted with non-specific proteins

  • Ensure primary antibodies have been purified in an appropriate way. For polyclonal antibodies these should be immune-affinity purified to the immunogen whereas monoclonal antibodies can be purified with protein A/G.

Incomplete blocking of membrane

  • Increase blocking incubation time
  • Increase the concentration of blocking buffer

Target is at lower MW than expected

Target protein has been subject to proteolytic cleavage

  • Try including protease inhibitors to the lysis buffer when preparing samples.
  • Make sure samples are prepared on ice
  • Try using a fresh sample

Target protein has been subject to proteolytic cleavage

  • Try including protease inhibitors to the lysis buffer when preparing samples.
  • Make sure samples are prepared on ice
  • Try using a fresh sample

Antibody epitope is present in another protein

  • Ensure a negative control sample has been included.
  • Try a different antibody using a different epitope
  • Decide if the extra band is an issue for the planned experiment.

Target is at a higher MW than expected

Target protein may have been subject to post-translational modifications such as glycosylation

  • Check literature for previous reports of post translational modification in the target
  • Attempt to remove the post-translational modification using enzymes such as deglycosylases. 

Target protein may have formed dimers or multimers

  • Try increasing the concentration of β-mercaptoethanol in the sample buffer
  • Try using stronger reducing agents

 

8. Further reading

Bell, 2016. Quantifying western blots: none more black. BMC Biology; 114: 116

Gassmann et al, 2009. Quantifying Western blots: Pitfalls of densitometry. Electrophoresis 2009; 30: 1845-1855

Pillai-Kastoori et al, 2020. A systematic approach to quantitative Western blot analysis. Analytical Biochemistry; 593: 113608