Sample Processing is to collect target protein. The processing shall be done as gently as possible as protein denatures or degrades easily. It is also very important to store processed sample, especially avoiding repeated free-thaw cycles. Processed sample can be sub-packed and sealed. Expiry Time: a week at 4 degree; a month at -20 degree; two months at -80 degree. Just put sample in room until its temperature reaches room temperature before using the sample. It is forbidden to heat the sample to melt the sample.
1. Use 3 vials T25 or 1 vial T75 (cell amount 1x107), and collect suspending cells. Or, scrape anchorage-dependent cell and then collect it.
2.Put sample in centrifuge tube and then set at 4 degree and centrifuge it at 2500rpm for 5 minutes.
3.Take supernatant and test it. If there is sediment, then add pre-cooling PBS and blow and mix it evenly. Also set at 4 degree and centrifuge it at 2500rpm for 5 minutes.
4.Abandon supernatant, and then add proper cell lysis buffer (RIPA, including PMSF). Later put it on ice and split it for half an hour or an hour. During splitting, it is better blow or vibrate centrifuge tube to split protein completely.
5.Set at 4 degree and centrifuge it at 10000rpm for 10 minutes.
6.Take supernatant into EP tube and store it at -80 degree