Western blotting (WB) Guide and Troubleshooting

Jump-to-Sections:

Overview:

- Western blotting
- Western blotting protocol
Methods/Protocols:
 - Sample preparation
 - Gel-electrophoresis
 - Transfer of proteins
 - Immunological detection
 - Controls
Troubleshooting:
 - No bands or weak signal
 - High background
 - Multiple/extra bands
 - Uneven/patchy spots on blot
 - Curved effect of the bands
References:
Related Protocols:
 - Deglycosylation of sample
 


(Download PDF)

Overview:

Western blotting

Western blotting is a laboratory technique used by life science researchers and diagnostic laboratories to detect specific proteins within a homogenate or extract of a biological sample.  Before immunological detection, sample proteins must be extracted from their source using an optimized lysis buffer, separated within a gel using an electrophoresis unit and transferred to a membrane via a “blotting” procedure.  Proteins can be separated within a gel by the size of their individual polypeptide subunits to determine their molecular weight, or they can be separated based on their native, tertiary form such that subunit interaction and conformation are preserved.  The latter form of separation is determined by their charge-to-mass ratio.  After the proteins have been immobilized on the membrane, the proteins of interest can be identified through immunological detection using antibodies conjugated for visualization via chromogenic, chemiluminescent, fluorescent or radioactive mechanisms.

[back to top]

Western blotting protocol

1. Obtain sample and extract targeted proteins.
2. Prepare reagents and samples for gel-electrophoresis.
         •    Samples in sample-loading buffer
         •    Running buffer
         •    Gel
         •    Standard
3. Load Molecular Weight Marker, samples and prepared Standard in the wells of the gel and run according to manufacturers’ instructions.
4. Assemble gel in electrophoresis unit and turn on the power unit.
5. While the gel is running, prepare reagents and materials for transfer.
         •    Membrane
         •    Filter Paper
         •    Transfer-buffer
6. Assemble gel and membrane within the blotting unit and transfer proteins onto the membrane according to manufacturers’ instructions.
7. Prepare reagents for immunodetection.
         •    Primary antibody solution
         •    Blocking buffers
         •    Wash buffers
         •    Secondary antibody solution
         •    Substrate solution
8. Remove the membrane from the transfer system and place in a container with Block Buffer.
9. Pour off the Block Buffer and pour the previously prepared primary antibody solution onto the membrane and rock at room temperature for 1 hour.
10. Pour off the primary antibody solution and add Wash Buffer and rock at room temperature for 5 minutes. Repeat this process 2 more times for a total of 3 washes.
11. Pour off the third wash and add the secondary antibody solution. Incubate with rocking for 1 hour at room temperature.
12. Pour off the secondary antibody solution and add Wash Buffer and rock at RT for 5 minutes. Repeat this process 2 more times for a total of 3 washes.
13. Pour off the Wash Buffer and add prepared substrate solution and rock for 5 minutes or until sufficient color has developed.
14. Wash the membrane to stop the reaction.
15. Visualize the membrane while it is wet and then allow to air dry.

[back to top]

Methods/Protocols:

Sample Preparation:

The procedure to extract and isolate proteins in preparation for Western blotting is dependent on multiple factors:

  • Tissue or cell type (animal, plant, yeast, bacteria, insect and/or fungi)
  • The location/properties of targeted proteins (cytoplasmic, membrane-bound, nuclear, mitochondrial or whole cell), and
  • The ability of your antibody to recognize antigen (native or non-native). 

These factors must be considered and will influence the equipment and/or lysis buffer you use to extract/isolate your targeted proteins. 

Animal tissues usually require both mechanical and detergent lysis because of the complexity and density of the tissue, while cells obtained from cell culture may only need detergent lysis.  Detergents are used because of their ability to break the lipid barrier and solubilize lipid/lipid, lipid/protein and protein-protein interactions.  This innate ability is due to the detergents hydrophobic and hydrophilic structure.  Extracting proteins from plant cells can also be difficult because of their rigid cell wall.  Bacterial cell types are the easiest to extract proteins from.

When isolating proteins of a specific region within a cell type, specific detergent types can be used.  Detergents are graded on their critical micelle concentration (CMC), which is determined by how many detergent monomers it takes to aggregate a single micelle.  For whole cell, nuclear, mitochondria or membrane bound proteins it’s common to use a detergent such as NP-40 or a RIPA buffer composed of multiple detergents.  Cytoplasmic proteins bound to the cytoskeleton may be extracted using Tris-Triton and soluble cytoplasmic proteins may be extracted using Tris-HCl. 

A major difference between lysis buffers is the inclusion of reagents that will cause proteins to denature.  For this reason, it’s important to understand the compatibility of your primary antibody to recognize the antigen in its native or non-native state.  This way you can choose the appropriate lysis buffer reagents.  An antibody that can only recognize the native form of your targeted protein may not be compatible to detect the protein in its non-native, denatured form.  Denaturing/solubilizing extraction buffers commonly contain an ionic detergent called sodium dodecyl sulfate (SDS) that wraps around the polypeptide backbone to dissociate it into its respective subunits.  Other detergents include sodium deoxycholate, sodium cholate, triton-X, and NP-40.  When it’s necessary to isolate proteins in their native form, a mild non-ionic detergent should be used, without SDS.  

It’s also important to choose a buffer system that will avoid protein degradation within your sample.  Using protease or phosphatase inhibitors can protect against serine proteases, cysteine proteases, metalloproteases and aspartic proteases.  Additionally, you can minimize protein degradation when collecting your sample by snap-freezing it in liquid nitrogen or starting protein extraction as soon as the normal biological environment of the sample has been compromised.

Along with your buffer a physical extraction/solubilization procedure can be implemented to extract your proteins of interest.  This can be done using a mortar and pestle, blender, homogenizer or by sonication.  The mortar and pestle method is a common technique used to crush snap-frozen samples before the extraction step, while liquid homogenization is used on fresh/thawed samples. Sonication uses pulsed, high frequency sound waves to dissociate samples. 

The final step in preparing your samples will be to determine the total protein concentration of your lysate to accurately load your samples into the gel for electrophoresis.  Popular assays for determining protein concentration include the Bradford method, Lowry protein assay, and the Bicinchonic acid (BCA) protein assay.

[back to top]

Gel-electrophoresis

Gel-electrophoresis is a technique used to separate proteins via an electrical current.  Proteins are commonly separated using polyacrylamide gel electrophoresis (PAGE) as an analytical tool to provide information on the charge, mass, purity or abundance of protein.  Proteins can be separated according to their physical properties such as charge-to-mass ratio (Native PAGE), mass (SDS-PAGE) or both isoelectric point and mass (2-D PAGE).  In native PAGE, electrophoresis is based on proteins charge to mass ratio because migration occurs based on the individual proteins net negative charge and their size/shape.  In SDS-PAGE, separation of proteins is achieved solely according to mass because the reagent sodium dodecyl sulfate (SDS) is used to give an equal charge to all proteins.  In 2-D PAGE, proteins are separated first according to their isoelectric point and second according their mass allowing for the best resolution of protein.  

There are a variety of gel attributes that need to be considered to optimize the migration of proteins within the gel.  The percentage of acrylamide within the gel, the thickness of the gel and the gel size can all influence the proteins within a run.  As you increase the concentration of acrylamide used to construct the gel, the pores within the gel will become smaller.  Using a lower percentage gel will be more ideal to resolve large proteins and higher percentage gels are more ideal for smaller proteins.   How thick your gel is will also affect the transfer of proteins from the gel to the membrane during the “blotting” step.  Proteins are easier to transfer to a membrane when using a thinner gel than a thicker gel.  If your sample volume will allow, use a thinner gel.  If your sample has a wide range of protein sizes, a gradient gel can be used such that the gel matrix is composed of varying percentages of acrylamide to capture the most proteins possible.  

Other factors to consider during electrophoresis are the running buffer and the sample-loading buffer.  Often manufacturers will recommend buffer systems to use if you have purchased their pre-cast gel.  Running and sample buffers are dependent on the type of gel you are using, your electrophoresis equipment, whether or not you are denaturing the proteins, reducing disulfide bonds, optimizing the pH, or visualizing your proteins during electrophoresis.  If you are running denatured proteins, your buffer system may contain SDS.  2-Mercaptoethanol can also be used in your sample-loading buffer to reduce disulfide bonds, ensuring denatured conditions of your sample.  Bromophenol Blue is used in sample-loading buffers to visibly monitor the process of agarose gel-electrophoresis.

After completing gel-electrophoresis there are numerous ways to visually detect your proteins in the gel.  Some of these include autoradiography, zinc staining, silver staining, coomassie dyes, and fluorescent staining.  The most common method used is the coomassie stain, which binds non-reversibly to proteins.  Overall, the type of stain you choose will depend on your downstream application to minimize any chance of interference.

[back to top]

Transfer of Proteins:

Once proteins have been separated within a gel, they must be transferred and immobilized on a membrane for immunological detection.  A nitrocellulose or polyvinylidene difluoride (PVDF) membrane is commonly used for the immobilization of proteins because of their hydrophobic binding properties.  There are advantages and disadvantages of each.  Nitrocellulose membranes form weaker bonds with transferred proteins, while PVDF membranes form stronger bonds that will tolerate more SDS and re-probing during labeling.  PVDF membranes are also more expensive than nitrocellulose membranes and require a pre-treatment step with methanol. 

There are different systems for transferring proteins to a membrane, but most common today are electroblotting systems that use an electric current to pull negatively charged proteins from the gel into the membrane traveling towards the positively charged electrode.  These systems can be wet-transfer or semi-dry transfer systems depending on the equipment used.  It’s also possible to pull proteins from a gel onto a membrane using methods that rely on capillary action and not on an electrical current. 

To transfer the proteins from the gel to the membrane, a “sandwich” is made comprised of the gel, membrane, and filter paper.  It’s important to properly secure the gel and membrane between the filter paper to ensure proper contact is made.  If air bubbles get between the gel and the membrane, a poor/uneven transfer will be the result.  Depending on your transfer system, the manufacturer will illustrate the proper assembly of the gel/membrane/filter sandwich.

A quality transfer can be confirmed using the Ponceau Red dye.  Ponceau Red is a reversible staining solution allowing you to visualize the transferred proteins and then rinse away the stain for subsequent immunological detection.  A quality transfer will have sharp, distinct protein bands.  A patchy or uneven appearance indicates a poor transfer, probably caused by insufficient contact between the membrane and gel.

[back to top]

Immunological detection:

Immunological detection is the process of incubating the PVDF or nitrocellulose membrane with antibodies to form the antigen-antibody complex for the detection of the targeted protein.  
Before incubating the membrane with antibodies, the membrane should be incubated with blocking agents to minimize non-specific binding of the antibodies to the membrane.  The blocking agents should not disrupt your proteins of interest from the membrane.  Bovine serum albumin (BSA), non-fat milk, casein, gelatin and tween-20 detergent are all common blocking reagents.  You should verify the compatibility of your blocking reagent with the detection reagents.  For example, non-fat milk cannot be used with biotinylated antibodies because milk contains biotin that can cause non-specific binding.

After blocking you will incubate your membrane with antibodies for immunological detection.  There are multiple labeling techniques including direct, indirect and indirect with signal amplification.  Direct labeling uses a primary antibody directly conjugated to a signaling source, while indirect labeling has a signal generating secondary antibody that will attach to the primary antibody making contact with the antigen.  For example, a primary antibody produced from rabbit would be detected using a secondary antibody that is anti-rabbit and conjugated to a detecting probe.  Finally, the indirect with signal amplification technique is accomplished using a biotinylated secondary antibody and an amplification reagent such as streptavidin.  

Detection is commonly achieved using a colorimetric (enzyme) reaction or a fluorescent signal generated from an ultraviolet source.  Immunofluorescence techniques use antibodies chemically conjugated to fluorescent dyes that are visible under a UV light.  Two popular dyes are Fluorescein Isothiocyanate (FITC) and Tetramethyl Rhodamine Isothiocyanate (TRITC).   Colorimetric reactions use antibodies chemically conjugated to the enzymes horseradish peroxidase or alkaline phosphatase.  The enzyme catalyzes the reaction of the substrate forming a color precipitate.  Common substrates include Diaminobenzidine (DAB), 3-Amino-9-Ethylcarbazole (AEC), 4-Chloro-1-Naphthol (4-CN) and Bromochloroindolyphosphate/Nitro Blue Tetrazolium (BCIP/NBT).

[back to top]

Controls:

Controls are a necessary component of Western blotting.  Use a positive control to ensure that the antibody recognizes the targeted protein and that your protocol is setup correctly.  Manufacturers will usually supply a positive control lysate with a primary antibody or specify what type of sample to use as a control.  As a negative control, load your loading buffer without sample to very immunodetection and non-specific binding does not occur.  

It’s also necessary to control the amount of sample you load in each lane of the gel and the transfer of sample from the gel to the membrane.  This is called the loading control.  Loading controls are a key part of studies comparing the expression level of proteins in different samples.  Common loading controls include Beta Actin, ERK, GAPDH and Tubulin.

[back to top]

Troubleshooting:

Common problems encountered include no bands/weak signal, high background, multiple/extra bands, patchy spots on the blot and curved effect of the bands.

 No bands or weak signal:

Problem Possible Solution(s)
Not enough antibody.
 Increase the concentration of primary or secondary antibody, or increase time of incubation.
Too little protein.
 Increase the amount of protein loaded per lane.  Verify solubilization and extraction protocol used on biological sample.
Problem with Transfer.
 Visibly check for proper transfer using the reversible stain Ponceau S (red).  Check electrical source and verify the transfer was performed in the right direction.  Optimize the transfer time and voltage conditions.  Ensure proper contact was established between membrane and gel.
Antibodies are incompatible.
 Verify primary and secondary antibody are compatible.
Antibodies inactive.
 Prepare fresh antibody dilutions and use proper storage technique as recommended by manufacturer.
Sodium Azide inactivates horseradish peroxidase (HRP) labeled antibodies.  Avoid using Sodium Azide or choose a different labeling mechanism.
 Bacterial contamination diminishes activity of HRP labeled antibodies.  Ensure proper storage and use sterile techniques when handling.

[back to top]

 High background

Problem Possible Solution(s)
Non-specific binding of primary and secondary antibody. 
 Lower the concentration of primary antibody.  Verify secondary antibody compatibility - try no primary antibody control.  Increase blocking incubation period.
Insufficient wash procedure.
 Increase the amount of wash steps to minimize unbound antibodies.
Membrane dried during incubation.
 Cover to ensure proper hydration.
Ineraction with non-fat dry milk. Use bovine serum albumin for blocking incubation step to avoid milk's endogenous biotin and phosphoprotein casein.

[back to top]

Multiple/extra bands

Problem Possible Solution(s)
Presence of post-translational modifications with higher molecular weight proteins.
 Investigate possibility of phosphorylation, glycosylation, acetylation, methylation or myristylation.
Too much protein loaded per lane.
 Decrease/optimize total protein loaded.
Sample degredation.
 Use fresh prepared samples.  Optimize the use of protease/phosphatase inhibitors in your sample buffer.
Non-specific binding of secondary antibody.
 Use no primary antibody control.
Primary antibody concentration to high. Decrease contamination.  If possible, use affinity purified antibody.

[back to top]

Uneven/patchy spots on blot

Problem Possible Solution(s)
Insufficient contact during transfer may have caused bubbles/air between the gel and membrane.
 Ensure proper gel-to-membrane contact during transfer.
Too little wash solution.
 Verify membrane and filter paper is fully immersed during wash steps.
Bad reagents.
 Make fresh reagents.

[back to top]


Curved effect of the bands

Problem Possible Solution(s)
Migration was too fast and/or hot. 
 Slow down migration by decreasing the voltage.  Reduce the temperature during electrophoresis using an ice bath or place the unit inside a cold room.

[back to top]


References:

1. McNamee MG. (1989) Isolation and characterization of cell membranes. Biotechniques 7(5):466-75.

2. Findlay JBC, Evans WH. (1987) Biological Membranes: A Practical Approach. IRL Press, Oxford.

3. Harlow E, and Lane D. (1999) Using Antibodies. Cold Spring Harbor Laboratory Press, New York.

4. Hames BD and Rickwood D. (1998) Gel Electrophoresis of Proteins: A Practical Approach 3rd Edition, Practical Approach Series, Oxford University Press.

[back to top]


Related Protocols:


Deglycosylation of samples:


1. 10 mL sample
2. 1.5 mL 10X Tris acetate buffer
3. 2 mL Chondroitinase (1x10-3 U/mL)
4. 2 mL Keratanase I (1x10-3 U/mL)
5. 1 mL Keratanase II (1x10-4 U/mL).
6. Incubate at 37 °C overnight.
7. Dilute sample 1:1 with 2X SDS reducing buffer

[back to top]