How many cells to lyse for Western? - Decreasing signal in my Western Blot (Feb/09/2006 )
How many cells should I lyse for a western Bot? I lysed 3million cells in 3 mLs of LDS sample buffer. I have a weak signal. Did I lyse in too much volume???
You may want to determine how much Total Protein you have in your sample via a Bradord Assay etc. You should be looking at loading 10-40ug total protein in each lane for good detection.
To me it sounds like you are lysing in too much buffer as I use 100ul for a 6-well plate (seeded with 5x105 cells) This is equivalent to you lysing in 5x the volume i use. But once again.. even if you have suspended in too much volume you just need to load more on your gel and you can work out how much by doing a Bradford. Unfortunately if you need to load more than 30-40ul (or what can fit into your gel wells) to load less than 10ug TP you will still gain a weak signal and have to prep your cells again.
Hope it works out for you.
In my experience, LDS buffer has not been used for lysis. I personally use RIPA buffer (1mL/1x10^7 cells) supplemented with freshly added protease inhibitors. I subesquently rock my lysate at 4deg C for 30 min and centrifuge at 15,000xg and 4deg C for 15 min. Run a Bradford assay to determine protein concentration, then add LDS buffer (+reducing agent?!) and heat. Perform SDS-PAGE on varying concentrations of lysate and probe with a constant concentration of primary antibody.
It sounds like you've diluted you lysate too much. Try 1mL RIPA/10^7 cells.