Molecular Weight Prediction? - (Mar/06/2006 )
Hello,
In cooperation with a company we designed some antibodies against a certain membrane receptor. The problem is that after Western blotting a band is generated with a molecular weight that is +- 40 kDa too high.
Theoretical weight = +- 42 kDa
Observed weight after western blotting = +- 80 kDa
I thought that some post-translational modifications might have occured...
What I am searching for is a MW calculator that takes into account any post-translational modifiction that might have occured. In this way I know that this band that I see is possible or not.
Can anyone help me out?
Thnx in advance
Are you sure your protein is fully denatured? If you are running a denaturing gel but your protein requires additional conditions like antioxidant or reducing agents then it may have partially renatured (e.g., reformed disulphide bridges, etc). If that is the case then it will not migrate as expected and might end up migrating slower thus giving the impression of a larger molecule.
Of course if you are running a native gel then you might be looking at a dimer or two different forms of the protein that are structurally distinct.
Check the literature to see if there are special conditions needed to run your protein on SDS-PAGE
could it be a dimer of the original protein?
Seb
Also, how are you estimating size? If you're using pre-stained (colored) markers, that could be part of the problem. Markers with dyes attached run at different relative MWs depending on conditions (esp. pH), and the difference could easily account for the 40 kDa you're seeing.
See, for example, page two of the product insert for Invitrogen's prestained markers here.
First of all ... thanks for the fast replies 
I've never thought of it that my protein could be only partially denaturated (I'm running a denaturating gel). I'll look into that by reboiling my samples and rapidly cooling them on ice before loading on the gel.
I'm estimating my size using a marker which gives blue bands. The marker that I use is the following: "prestained SDS-PAGE standard, high range" from BioRad cat. 161-0309
The protein I'm investigating is a receptor protein, so it could also be a dimer that I'm seeing. The only problem is, how do you know that for sure?
What's also weird, is that other proteins appear at the correct size on my gel. For example beta-actin (that I use to ensure equal loading in all my slots) appears at +- 42 KDa which is the correct size. Is it possible that some proteins are fully denaturated and others are not?
Does everybody here rule out that this phenomenon may be due to posttranslational modifications (phosphorylations, glycosilations,...)?
questions ... questions ... questions
Is your protein a well-known receptor? If so then there should be plenty of literature about possible post-translational modifications.
As for renaturation, don't just reboil but investigate the use of antioxidant or reducing agents. Some proteins require these to maintain disruption of disulphide bridges. Also some protein should not be boiled at all before loading as it can cause oligomerisation or aggregation. One example of this is P-glycoprotein which should simply be added with reducing buffer at RT.
I can't say I know of the top of my head what PTMs might increase Mw by as much as 40 kDa. However, it could be possible, see link below where the predicted mass of a receptor was 20-30 kDa lower than observed.
http://www.ncbi.nlm.nih.gov/entrez/query.f...8&dopt=Abstract
Literature searches might be your best bet if the gel alterations yield nothing