Troubleshooting Western Blots

Multiple bands are detected

The detection of multiple bands is not necessarily cause for alarm. In some cases these bands may represent specific reactivities with alternatively spliced isoforms of an antigen that happen to possess common epitopes. Alternatively, additional bands may represent non-specific immunoreactivity with seemingly unrelated proteins. Therefore, when multiple bands are observed, it is necessary to determine if this reactivity is antigen specific or the result of non-specific reactivity and whether this non-specific reactivity is attributable to the primary or secondary antibody. Often, specific immunoreactivity is determined by employing a preabsorption control. The source of non-specific reactivity can often be elucidated by performing a secondary antibody only control.

Apparently specific bands of unanticipated molecular weight are observed

Typically this is due to proteolytic degradation of the protein sample or incomplete reduction of sample prior to electrophoresis. Multiple bands migrating near the expected molecular weight of the antigen is often characteristic of incomplete reduction. Proteolytic degradation of the sample should be suspected if all the observed bands are of lower molecular weight than anticipated.

No bands are observed

Some antibodies, particularly monoclonal antibodies generated against native antigen, may not recognize proteins that have been fractionated under reduced and/or denatured conditions. Such antibodies are thought to recognize conformational epitopes that exist by virtue of the native three dimensional structure of the antigen that is lost upon denaturation and/or reduction. In such cases, the reducing agent may need to be eliminated or gels may need to be run under non-denaturing conditions (w/o SDS). Finally, if using peroxidase conjugated secondary antibodies, all solutions must be free of sodium azide because even trace amounts of azide will inhibit the peroxidase activity, leading to no bands being observed on the blot when it (or the film) is developed.

SDS-PAGE gel did not run correctly

Electrophoresis of proteins in a reducing SDS-Polyacrylamide gel provides only an estimate of protein size, not an exact measure. This is due to several factors that affect proteins’ mobility through the gel.

Conceptually, SDS-PAGE electrophoresis works as follows. The protein sample is mixed with a sample buffer that generally contains a reducing agent, a buffer, glycerol and SDS. Usually the reducing agent is either dithiothreitol (DTT) or 2-mercaptoethanol (BME). The function of the reducing agent is to disrupt disulfide bonds present between cysteine residues in the protein. This disruption can also be enhanced by heating the sample prior to loading. Sodium dodecyl sulfate (SDS) an anionic detergent, denatures secondary and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass. The SDS binds to the protein in a ratio of approximately 1.4 g SDS per 1.0 g protein (although binding ratios can vary from 1.1-2.2 g SDS/g protein), giving an approximately uniform mass:charge ratio for most proteins. This results in migration of the protein through the gel can be assumed to be directly related to the size of the protein.

Several factors can affect the migration of proteins and cause them to migrate at a slightly different rate than predicted based solely on its Molecular Weight (MW).

1. Incomplete reduction of the sample: incomplete reduction is often characterized by the presence of multiple bands at and around the predicted size of the protein. Add fresh reducing agent and re-run sample.
2. Differences in SDS binding: The amino acid composition of each protein is unique, and the different side chains present on the individual amino acids cause each protein to bind SDS with varying affinity. (Different proteins bind from 1.1-2.2 grams of SDS pre gram of protein). This difference in binding can cause significant differences in the actual mobility of the protein compared to the predicted.
3. Degradation of the protein sample: Often bands at less than the predicted size are the result of degradation of the protein sample prior to electrophoresis. Use fresh lysates containing protease inhibitors that have been properly prepared and stored.
4. Inappropriate polyacrylamide concentration: Samples of specific molecular weights migrate best in certain percentage SDS-PAGE gels, choice of gel effect protein migration. (See: “What percentage gel should I run?” in knowledge base.)
5. Migration of Molecular Weight Markers (MWM): there are many different MWMs available, some of which may not be optimal for predicting the mass of the protein in the sample (i.e. too large or too small). The use of pre-stained molecular weight markers is very helpful for assessing transfer of proteins in immunoblot applications however, pre-stained markers may migrate at sizes slightly different from predicted due to the presence of attached dye molecules.

While this list is by no means all inclusive it does provide a guide to why a protein of interest may migrate at distances different than the predicted in SDS-PAGE electrophoresis. While an extremely useful and powerful tool, SDS-PAGE is by no means predictive of accurate protein molecular weight.