protein showing up around 50 kda rather than 20 kda - (Jul/17/2012 )
Dear forum contributors,
I have samples saved in RIPA with PI of MSSA and every time I run my western blot I get very clear bands at around 50kda rather than 20kda. I have stained my gel with coomasie blue to make sure the proteins are coming off the gel. I've used pseudomonas as a negative control (the positive control is too expensive for us to buy). We've varied our transfer times to PVDF from 15-30 minutes and varied our film exposure times. We've bought new ECL and Magic Mark XP (the magic mark doesn't seem to show up very much at all, but See Blue is working just fine). I've added extra BME even though it is already in my laemelli buffer (to make sure there aren't dimers). Next, I am going to add DTT (to check for dimers again). I always centrifuge the samples at 8000xg to get rid of the membrane pellet and boil the samples for 5 minutes before loading them in the gel. I tried boiling for an extra 5 minutes last time. We are using sheep anti-staphylococcus alpha-hemolysin antibody and donkey anti-sheep with HRP as the secondary antibody.
After I use DTT this week, I will be out of ideas. By the way, I am kind of new to my lab and my PI said our lab has had problems with proteins showing up as too large. Any ideas?
Amy in Kentucky
The first thing I would suspect (after dimers) would be that the antibody is non-specific. If you can find some peptide against the antibody epitope and perform an antibody competition experiment to confirm the specificity, that would be best.
You could also get people to streak their nasal flora on a mannitol salt plate to look for Staph aureus, which is a common producer of alpha-haemolysin - about 30% of people carry Staph a, and some of those are bound to be positive for the haemolysin - which you could then use as a positive control.
Thank you for this post. You are right. Our primary antibody is polyclonal, but according to the woman that is teaching me how to troubleshoot this, there is no monoclonal antibody for alpha-hemolysin that is available. This is helpful, thank you!