The above answers are quite possibly right.

But you may also want to check for post-translational modifications of your ABC protein.

There are reports of ABCs being phosphorylated and glycosylated. If this is the case for your protein, the predicted size can be a lot lower than the size you observe.

You can try multiple approaches to asses this. Including Immunoprecipitating your HA protein and then treating with a non-specific phosphatase like alkaline phosphatase to remove phosphates. or treating with PNGaseF which will deglycosylate you protein. you may well then see a shift down to a MW that is closer to your predicted size.

It looks like you've done a thorough job checking your constructs. You might want to check your size standards if they're prestained. The SDS-PAGE gel type can affect their migration. For example See Blue Plus Prestained standard Myosin protein migrates at 250kDa in Tris-Glycine gels but at 188kDa in NuPAGE MES gels. Some company's prestained standards are very inaccurate at high molecular weight in some gel percentages as well. If you pour your own SDS-PAGE gels, the premarked standards will not run as advertised. You can recalibrate the prestained standards in your gel system by running lanes of nonstained standards beside stained standards and coomassie staining the gel. The nonstained standards are the real molecular weights as they're not modified by dyes.

I agree with Jeff, in addition, proteins with long regions of positive or negative charge can sometimes run strangely on a gel. I believe that sometimes proteins with negative patches can migrate more slowly because they do not bind SDS as efficiently, so maybe you can try heating it with your loading buffer for a longer time.

The above answers are quite possibly right.

But you may also want to check for post-translational modifications of your ABC protein.

There are reports of ABCs being phosphorylated and glycosylated. If this is the case for your protein, the predicted size can be a lot lower than the size you observe.

You can try multiple approaches to asses this. Including Immunoprecipitating your HA protein and then treating with a non-specific phosphatase like alkaline phosphatase to remove phosphates. or treating with PNGaseF which will deglycosylate you protein. you may well then see a shift down to a MW that is closer to your predicted size.

I totally agree with Neil. My first idea was if you had considered post-translational modifications. Are there any signal peptides, phosphorylation or glycosylation sites predicted?

Once I was working with plant-specific protein with expected MW of 33kDa. However, it was always showing up at 55 kDa on Western Blot after over expression in plant cell culture. When I treated the extracts or purified protein with CIAP, it got back to 35kDa on SDS-PAGE and WB. Try to remove PTMs. Thumbs up to Goetz and Neil!

Is the protein you cloned a membrane protein (ie an ABC transporter)? Then you should also consider that membrane proteins often tend to migrate differently in an SDS-PAGE gel compared to globular proteins (such as those in your size standards) . But I also agree that checking for post-translational modifications is a good thing to do. Good luck!

I agree with all the above, and think you should focus first on the type of gel you are running.

did you check that your antibody did not react with untransfected cells?

That would require you to do something to remove the unspecific signal then your protein would be detectable.

Otherwise if it is a membrane protein you could try to not boil you samples or use lower temperatures, it helps.

I really like the answers given by all these guys...good observations!!

one more possibility..that I can think of is..the size of ur protein seems to be little higher than 250 kDa. Try to run 9-10% SDS PAGE and over-run the gel such that your protein comes a little down the gel. if the size is > 350kDa....ur protein is forming dimer. ABC transporters are known to form dimers. just a rare possibility. try it after performing the analysis for PTMs- phosphorylation/glycosylation.

Post-translational modifications in which case migration is not directly related...check for this possibility in prosite...maybe it not a problem and it's just something interesting that gives you some new questions to answer. Mass spec will give you the answer.

Hi, Satadru. Did you purified your protein? You can do the small scale purification (for example use Pierce Anti-HA Agarose) and Mass spec, with such data you will see where to move with your experiments.

I agree that the observation is possibly due to post-translational modification. If this is the case, you will obtain smaller size of band on gel (close to expected size) if the protein is over expressed in E coli cells, which has limited capacity for post-translational modification campared to mammalian cells.

Have you cloned your protein with any tag? If you cloned your ABC protein with any tag, you should calculate tag + ABC protein. You also use some software and find out the exact mol. wt after post translational modification. You also try in native gel instead of SDS-PAGE and find out any dimer or not.

Good answers already! One more thing I would like to add to Andaleeb: If you are looking for dimerisation than compare one sample with loading dye w/o DTT or beta-ME against a sample with DTT/beta-ME and boiled for 3 minutes. This can show you if there are disulfide bonds between your protein dimers, because the sample with dtt/beta-ME would remove this connection and the one w/o would not. Good luck!

It's not a dimer. It doesn't add up and you would see some monomer band, and there is no trace of a monomer band in your example. It's glycosylated. See endless examples of other ABC transporters... P-glycoprotein, CFTR, MRP, etc. The PNGase F experiment is your best bet. You will see a band more like what you expect and probably sharper too (glycoslyated proteins tend to give diffuse bands) after PNGase F treatment. Boiling large membrane proteins in SDS sample buffer is never a good idea (aggregation), better to incubate at 37 to 70 °C, temp. determined empirically.

Thank you for your reply....I will try to do PNGase F experiment....also I found out that I didn't mutate the start AUG of my ORF during cloning....that can be one of the reasons why I can't find the expression with anti-Tag antibody....translation may just begin from the ORF's AUG not the tag's !!