My protein is consistently resolving a lower molecular weight than expected. The expected size ~120kDa but I am detecting it at ~50kDa across multiple blots and cell lines. :/ Given that my control protein is detected at the expect size, I believe my samples are not degraded. I have attached a pic of blots.
Dear Mariana,
Are you sure that you have the totality of your protein? I think that you have just one part of your protein of interest (N-terminal domain or C-terminal,...). You can do some research in the litterature to know more about your protein (molecular weight of each region).
Did you do all steps of the protocol exactely like mentionned? Maybe the concentration of separation gel that you make is not appropiate to your protein of interest!!!
I think you need also to check the voltage applied and time of migration.
Hope this could provide an idea of what the cause might be.
All the best!
Hi Mariana, a 70k difference is quite substantial and can't be attributed to running conditions, especially if you have a control protein at the expected size.
The easiest explanation is that your protein is cleaved. Do you include proteinase inhibitors in your cell lysates?
The band you observe might be a parasite band produced by unspecific binding of your antibody. It would be good idea to run a positive control sample (some cells known to express your protein of interest). The suppliers usually tell you how the antibody was validated
Thank you all for your input!
I double-checked the %of my separating gel and 8% is appropriate for the size of my protein. I also use 1:100 ratio of proteinase inhibitor cocktail (Sigma) to RIPA buffer when preparing my lysates. I've looked into the literature and other researchers resolve the protein at ~120 as expected : / (though they are looking at stably transfected cell lines and not cells that endogenously express my protein of interest...not sure if that matters).
All my cell lines are known to express my protein of interest, I am trying to substantiate if the differential transcript expression I noted correlates to different amounts of functional protein. Please correct my understanding if it is wrong, but given that all my cell lines express my protein of interest they technically serve as my positive controls?
Also, how would I go about determining if non-specific binding is occurring?
Hi Alex Kashkin
, thank you for your detailed response. My blot is of EPHA8 (RTK) (actual expect size is 126kDa) it's involved in cell adhesion and migration according to the literature. I am trying to create a baseline transcript and protein expression profile of this family of RTKs, then I will do further downstream studies to explore their role in my cell lines. I would like to see if I alter the expression of this receptor, can I attenuate the invasive potential of my cells. But, first I need to show if an upregulation of transcript correlates to an increase in protein via my westerns.According to NCBI, there are at least two variants for EPHA8 (or two accession#s). Also, I referenced the data-sheet for my AB and there are multiple faint band detected on their gel, which suggests there are multiple cleaved variants, but none corresponding to ~50kDa. I have heard from other researchers that the company where I bought my AB has given others trouble in the past. So, if I get an new AB from another company and detect bands of ~126kDa then that will confirm my receptor is getting cleaved? But, how can I confidently determine if that what I am detecting on my blot is indeed the receptor I am probing for?
The protein you are trying to detect is localised in a membrane, they are notoriously difficult to disolve in a standard lysis buffer. I usually use 1% sds to detect membrane proteins and it works really well. Maybe try it and see if it helps
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