You mean that it was running at the expected size and all of a sudden it started exhibiting anomalous migration, or has it always run like that?

If the second, you should cut out the band and send it to an MS facility just to make sure everything is okay, but most likely there are no problems. Many proteins migrate slower or faster in SDS-PAGE than expected, owing to a variety of reasons: extreme pI (meaning, it will bind less or more SDS molecules per mass unit than usual), presence of proline-rich stretches (those confer rigidity to the peptide backbone), post-translational modifications (glycosylation, attachment of lipids, etc). If the protein has disulfide bridges, often omitting a reducing agent changes its relative migration (BSA is a classic example).

Hope it helps!

You mean that it was running at the expected size and all of a sudden it started exhibiting anomalous migration, or has it always run like that?

If the second, you should cut out the band and send it to an MS facility just to make sure everything is okay, but most likely there are no problems. Many proteins migrate slower or faster in SDS-PAGE than expected, owing to a variety of reasons: extreme pI (meaning, it will bind less or more SDS molecules per mass unit than usual), presence of proline-rich stretches (those confer rigidity to the peptide backbone), post-translational modifications (glycosylation, attachment of lipids, etc). If the protein has disulfide bridges, often omitting a reducing agent changes its relative migration (BSA is a classic example).

Hope it helps!

It would have been a lot more easier showing the picture of you gel or provide more details on procedure used for more clarifications. I am going to make several assumptions here. I assume you did western blotting using specific antisera to the targeted protein. Any band you get should literary indicate your protein of interest especially if there are no background bands which usually come from protein degradation.

On the other hand, I am thinking you stained your gel (with a dye e.g. Coomassie brilliant blue or other stains) and visualised it. If this is the case you may want to use a protein standard that have suitable range within 80-60kda.

Some induced conditions may contain proteins that interfere closely with the protein of interest (similar sizes). My work on protein expression of stress hormone-induced Burkholderia sp used serum as the treatment medium. The albumin in the serum was expressed at high levels close to out protein of interest.

Hope this help.

Purified proteins generally show anomalous migration. I had experienced this with one of my protein of interest. Protein migration in the SDS-PAGE is affected by the PI of the protein. In other cases, the anomalous migration becomes normal upon treatment of whole cell lysates with DNAase (1 ug/ml) plus RNase (0.2 ug/mL) for 30 minutes followed by heat denaturation at 95 degrees for at least 20 minutes. Hope it will help. gud luc

It could simply be the accuracy of the assay; bearing in mind it is measuring approximate molecular mass. Try using different gel gradients and, like Tombari Pius Monsi mentioned, use markers suitable for the gel and size of protein being analysed.

There are orthogonal methods you can use, for example Size Exclusion Chromatography, for size determination. Again, assay accuracy will be a factor.

The protein i work on is ~147kDa but I am happy if the size approximation is 145-155 kDa. The temperature and time of sample degradation plays a part as well as the detergent used (e.g. SDS v LDS).

And as mentioned by Alejandro Martin. Testing by MS is really the only sure way to accurately determine the mass. You can ship the sample for intact mass analysis as it is and also cut the band out of the gel for analysis. You'll be able to see a) whether the SDS-PAGE assay is creating an artefact and altering the migration (hence the ± 10% accuracy you're seeing) and b) what the actual mass of your protein in the formulation/buffer it is in.

Ravi Shankar Gautam , having a apparent molecular weight of 60kDa instead of the expected 53.5kDa should really not be a big concern. Molecular weights observed on gels are usually estimated and not exact values.

Hi Ravi,

As Robert said, u can also plot a log standard curve of yr band of interest and the protein marker migration pattern to actually be sure of the band size.

Hello Ravi,

I suggest you to try with different gel gradients and, like Tombari Pius Monsi mentioned, use markers suitable for the gel and size of protein being analysed.