I would like to do western blots to look at early and later events in TCR signaling such as Zap70 phosphorylation, Lck phosphorylation, PLCg phosphorylation, Erk p etc.. I see in papers that cells are typically incubated with the CD3 antibody on ice and then activated by crosslinking of antibody. People then look at very short time points like 2-5 mins as the 1st time point. I was wondering how these experiments are done for a western. Are the cells lysed directly in the media in which they are activated or are these cells actually spun down, washed and then lysed? If they are lysed directly, wouldnt this dilute the sample too much and wouldnt there be too little protein loaded per lane? But if they are spun down, washed and then lysed, how are the really short time points and intervals ensured?
I am new to immunology and would appreciate any help.
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