smearing in western blotting - (Aug/09/2011 )
I am using BioRad's gradient premade gels for my western blotting, but sometimes i get smears with my protein of interest (and sometimes i get a perfect sharp bands!). I don't see any smear with my ponceau, so i assumed my transfer was fine. Anyone has any input in this?
Ponceau is a lot less sensitive for specific proteins on the membrane, so you probably wouldn't see any smearing with the stain, but you will see it with the antibody based detection.
Smearing is often caused by adding too much protein and thereby overloading the gel.
Hi Bob, thanks for your advice, just a question though, if you said it's caused by too much protein, but why it doesn't happen all the time. I've always use the same amount of protein to load (based on Bradford assay) and same conditions, the only difference are just different samples. It's just confuses me
It depends on the sensitivity of the bradford assay - which has quite a narrow range where it is accurately measuring the protein content based on a standard curve. If you are outside this range then you are quite likely to get values that are apparently exactly the same, but in reality could be well over or under what you think you have.
Your protein sample contains, I am guessing, that is is a whole cell lysate? This contains more than just proteins. Lipid can cause this smear as well too.
Sometimes briefly centrifuging samples and remove the lipid layer helps.