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★ ★ ★ ★
小木虫(金币+0.5):给个红包，谢谢回帖
jhu132llh(金币+3, APEPI+1): good,感谢积极应助,很专业 2011-10-30 18:15:46
看看这个nature protocol
Sample extraction techniques for enhanced proteomic
analysis of plant tissues
我前几个星期，刚用这个protocol作的，还可以，你是要做2-D 吧。 主要是丙酮挥干这一步，不要太干，太干很难。 用超声可能能帮助溶解
步骤是这样的，和你的差不多，但少几步：
（1）Place frozen plant tissue into the cold mortar. Add acid-washed sand (0.5–5 g per 1 g fresh tissue; optional) and PVPP (1–15% (wt/wt); optional) and grind tissue into a fine powder using the pestle and mortar
（2）Suspend the powder in TCA extraction buffer (5-15 ml solution per gram tissue). Store at –20 1C overnight. Proteins should precipitate as white flakes
（3）Centrifuge the sample for 30 min at 5,000g at 4 1C. Carefully pipette out the supernatant and discard.
（4）Add 10 ml ice-cold acetone to the pellet and centrifuge the sample for 10 min at 5,000g at 4 1C. Carefully remove
the supernatant with a pipette and discard.
（5）Repeat Step （4）two more times
（6）Resuspend the pellet in a buffer for protein resuspension that is appropriate for the downstream analytical approach.
Notes：
Protein resolubilization is often one of the most challenging steps of the procedure. It may be necessary to increase the amount of buffer added to resuspend the pellet fully, and in some cases there may always be a residual pellet.
Pellets can be resuspended in a microcentrifuge tube with a mini-magnetic stirrer. Sonication can also be used to help resuspend the pellet, but care should be taken not to heat the sample, so it is recommended that the sample be sonicated in short bursts while dipping the tube in ice water. |
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