The Western blot assay provides valuable information about a protein including abundance, the apparent molecular mass, post-translational modifications and splice variants. However, analysis of the protein can be difficult if multiple bands appear on the blot. When dealing with multiple bands on Western blots, it is important to determine whether they are due to technical artifacts or whether they represent true variants of the protein of interest. This guide describes various causes of multiple bands due to technical artifacts and how to determine if multiple bands represent true variants of the protein of interest.
1. Multiple Bands Caused by Technical Artifacts.
Technical artifacts can cause the appearance of bands that have higher or lower molecular weights than the actual protein. The types of bands that are observed can help determine the cause of the artifact.
a. Lower and Higher Molecular Weight Bands
i. Concentration of Primary or Secondary Antibody too High
If the concentration of either the primary or secondary antibody is too high, the antibody can bind non-specifically to proteins other than the protein of interest. Non-specific binding can occur with any protein, thus bands of higher and lower molecular weight may be observed.
1. Titrate antibody
To determine if high antibody concentrations are resulting in multiple bands, titrate the antibodies. Both antibodies can be titrated simultaneously using a dot blot in a checkerboard like fashion (this will be linked to the protocol section on checkerboard titration).
2. No primary antibody control
To determine quickly whether the primary antibody is responsible for producing multiple bands on the blot, perform the entire Western blot procedure but omit the primary antibody. If multiple bands are still observed, then the secondary antibody is responsible for the artifacts.
ii. Excessive amount of lysate loaded on gel
If too much lysate is loaded onto a gel, antibodies can bind non-specifically to proteins of excessive abundance, resulting in multiple bands.
1. Decrease amount of lysate used
To determine the appropriate amount of lysate to load on the gel, load decreasing dilutions of lysate on the gel.
2. Increase washes
If the protein of interest is not very abundant, than it may be necessary to load large amounts of protein to detect the protein. To decrease non-specific binding to proteins of greater abundance increase the number and duration of washes.
iii. Antibody is “dirty”
Polyclonal sera or unpurified antibodies can also contain less abundant antibodies that bind to abundant, common cellular proteins.
1. Use affinity purified antibody
When possible, purchase affinity purified antibodies. Alternatively, polyclonal antisera can be affinity purified using immobilized Protein A, G or L to purify IgG molecules or immobilized antigen can be used to purify antigen-specific antibodies. Commercial kits are available for affinity purification of antibodies.
b. Lower Molecular Weight Bands
i. Degradation of target protein
Multiple lower molecular weight bands usually arise from poor handling of the lysates/samples prior to analysis and can be indicative of degradation of the sample.
1. Protease inhibitors
To decrease degradation of proteins by cellular proteases, include protease inhibitors in the lysis solution used for sample preparation.
2.Keep samples cold
Keep samples on ice during preparation and during all manipulations to limit protease activity.
3. Limit freeze/thaws
Aliquot samples into multiple tubes and freeze/thaw each tube only 1-2 times to limit degradation induced by temperature changes.
c. Higher Molecular Weight Bands
Higher molecular weight bands alone can also be due to technical artifacts.
i. Target protein may form multimers
Unresolved protein multimers can result in molecular weight bands that are higher than the predicted molecular weight of the target protein.
1. Boil in SDS-PAGE buffer for 10 minutes
To reduce proteins completely, boil the sample in SDS-PAGE buffer containing a denaturing agent such as dithiothreitol or β-mercaptoethanol for 10 minutes rather then the usual 5 minutes.
2. Compare reducing vs. non-reducing gel
To determine if the protein of interest is forming multimers, prepare the samples under reducing and non-reducing conditions prior to loading on the gel.
2. Determining Whether Multiple Bands are Scientifically Relevant
Multiple bands that are scientifically relevant can be observed during Western blot analysis. Higher molecular weight bands are seen when proteins become bound to modifiers whereas lower molecular weight bands are observed when proteins are spliced.
a. Determine whether bands are specifically recognized by the antibody
One of the criteria in determining whether multiple bands are due to technical artifacts or are scientifically relevant, is to determine whether the bands are specifically recognized by the primary antibody. This can be achieved by using control samples and/or by specifically inhibiting the interaction of the antibody with the protein.
i. Control samples
Include controls on the gel that contain or lack the protein of interest. For example, include cell lines that are identical in all respects except in expression of the protein to identify bands that appear due to non-specific interactions.
ii. Blocking peptides
When available, blocking peptides (those that match the epitope recognized by the antibody) can be used to determine which bands are specifically recognized by the antibody. Pre-incubate primary antibody with increasing concentrations of blocking peptides prior to incubation of the blot with the antibody. The blocking peptide will prevent interaction of the antibody with the protein target and the band will “disappear” on the blot.
b. Higher molecular weight bands
Higher molecular weight bands are often seen when the target protein is postranslationally modified. Acetylation, methylation, myristoylation, phosphorylation, glycosylation and ubiquitination are all modifications that increase the molecular weight of a protein. Several techniques can be used to determine if a protein is posttranslationally modified.
i. Protein analysis software
Use protein analysis software to predict the types of modifications a protein may acquire posttranslationally. Use the software to predict the molecular weight of the protein with and without modifications.
ii. Antibodies specific for modification
Commercially available primary antibodies are available that recognize specific posttranslational modifications (e.g. antibodies specific for tyrosine phosphorylation). These antibodies can be used to confirm posttranslational modifications. To use these antibodies, immunoprecipitate the protein of interest using the protein-specific primary antibody. Run the immunoprecipitated fraction on a gel and then perform a Western blot using the modification-specific primary antibody.
iii. Chemical treatments to remove modifications
Proteins and protein extracts can be treated with chemicals that will remove modifications (e.g. PNGase F is used to removed glycosylations). Complete removal of all modifications should result in a single band of the predicted molecular weight in Western blot analysis. When treating with chemicals to remove modifiers, it is important to also include non-treated samples on the same gel to observe the changes in the banding pattern.
c. Lower molecular weight bands
Lower molecular weight bands may be observed if additional forms of the protein of interest are generated through alternative splicing or cleavage of the protein posttranslationally. RNA and protein analysis software can be used to predict these events, however these modifications will have to be determined empirically for each protein.
i. Splice variants
Alternative splicing of an mRNA can generate multiple protein products. If the epitope that the antibody recognizes is shared between the proteins, then multiple bands will be observed.
ii. Protein is cleaved
Lower molecular weight bands might be observed if the protein is cleaved postranslationally. Polyclonal antibodies may recognize multiple lower molecular weight bands due to their ability to recognize multiple epitopes. Monoclonal antibodies will only recognize one band of the cleavage product(s).