cell culture western blot - (Oct/19/2006 )
you are right, a big band in the range of 66 to 67 kDa should be albumin; a disadvantage could be - this depends on your Ab - that large amounts of a protein on blot tend to bind some Ab´s unspecifically; you will be on the lucky side if you protein or proteins of interest are far from size of albumin
Hi. Thanks a lot for your helps. And another question: Can I freeze the lysate in -20 degree?
Yes, you can. Better at -80 but -20 is fine for a short period
-20°C for short term storage
-80°C for long-term storage
In any case: I recommend shock-freezing with liquid nitrogen to save biological activities of various enzymes and compounds!
Many thanks. I have another problem: my secondary AB is an HRP-linked. I think I can't use x-ray film. Then how can I quantify the bands.
Thanks in advance
HRP (horseradish peroxidase) linked secondaries are no problem. Just use a chemiluminescent reagent like the ECL from Pierce (no 32106) to create a luminescent signal you can expose to x-ray film.
As far as loading controls go, the best ones are generally structural proteins found in all cells--meaning things like tubulin or actin isoforms are excellent. I've personally used antibodies against beta-actin and alpha-tubulin as loading controls with great success in the past. Although, if you're looking at cell-free serum (not whole blood) you may be limited to using antibodies against albumin or sex-hormone binding globulin, or some other constitutively expressed serum component.