western膜蛋白的提取

分离膜蛋白的方法有两种：1先分离膜，然后提取；2用特殊的去污剂选择性的分离。第二种方法简单，可靠，但有时含有其他蛋白。

原理：4度时所有的蛋白质原则上都溶于TritonX114水溶液，但在37度时，此溶液分为水相和去污相；此时亲水性蛋白溶于水相，疏水的膜蛋白溶于去污剂相中。

方案

1）放射性标记受试细胞

2）将标记的细胞放在冰上

3）去除上清，用pH7。4的冷磷酸盐缓冲液洗涤单层细胞两次

4）加入1ml2%TritonX溶液冰浴15min

5)刮下单层细胞，4度下10 000g 5min离心

6）溶液37度水浴10min以分离水相和去污剂相，然后37度下2 000g离心5min

7)收集水相留作分析

8）用500ul冰冷的buffer C溶解去污剂相沉淀，冰浴2min后加温，在按步骤6再次离心

9）按步骤8再次抽提去污剂相，用buffer C将洗涤后的去污剂相稀释到初始体积

10）用等量的buffer A分别稀释水相与去污相，并进行免疫沉淀实验

试剂：

1）2%tritonX114:2%TritonX114、50mmol/L Tris HCl（pH7。5）、蛋白酶抑制剂

2）缓冲液A（含0。5mol/LNaCl的RIPA buffer)

3)buffer C

10mmol/L Tris HCl(pH7.5)

150mmol/L NaCl

5mmol/L EDTA(PH7.5)

这是我在nwfsc上求助膜蛋白提取时别人发给我的email，其实膜蛋白的提取的方法依据膜蛋白的不同类型方法也不一样。是组织还是细胞，细胞膜膜结合蛋白还是细胞器结构膜蛋白，如果是细胞的话建议你买个膜蛋白抽提试剂盒吧。

I'll give you these protocols that I use. The first is for isolation of mitochondria from liver and the other is for the isolation of membrane proteins. Both of these are specific to my needs and you will want to optimise them for your own needs. For the isolation of membrane proteins you will need to find references for your specific protein. The buffers and conditions that I use will not be suitable for all proteins.

Isolation of mitochondria (adapted from O'Gorman et al. FEBS Lett. 414 (1997) 253-257) Isolation buffer: Mannitol 200mM

Sucrose 70mM

HEPES 2mM

EDTA 0.5mM

BSA 0.5mM pH 7.4

Wash buffer: Mannitol 200mM

Sucrose 70mM

HEPES 2mM pH 7

All experimental work is carried out at 4degC and in the presence of 500uM PMSF. (the PMSF can be substituted for AEBSF but one or the other must be used)Liver is removed from Wistar rat and placed in cold isolation buffer. the liver is cut up into 4x2g pieces. Each piece is placed in

1ml isolation buffer and chopped up with scissors to form a mince. This mince is placed in a Potter tissue grinder and 2ml more isolation buffer is added. The mince is homogenised with four passes of the grinder and more isolation buffer is added until the total volume is 10ml. The homogenate is put into a centrifuge tube and 10ml more isolation buffer is added to give a 10% homogenate (2g of tissue in 20ml of buffer). Repeat this process with the other pieces of tissue.Differential centrifugation is used to isolate mitochondria.Centrifuge at 560g for 15min. This will give a pellet of large cell debris and nucleus (I discard this pellet)Centrifuge the supernatant from the first spin at 7000g for 15min. This gives a pellet of mitochondria. The supernatant will contain microsomes and cytosol (the microsomes can be separated by centrifuging at 21,000g for 40 min)Resuspend the pellet you want in 10ml of washing buffer and then centrifuge again at the speed required. Repeat this step until the pellet has been washed 3 times.Now you have a cellular pellet you can start to isolate your protein from it.With this protocol the most important thing to do is remember the AEBSF or PMSF. If you do not add it your protein may become degraded before you can isolate it.

Isolation of membrane bound protein (adapted from Zwizinski and Schmidt Arch. Biochem Biophys 294 (1992) 178-183 and Belzacq et al. Oncogene 20 (2001) 7579-7587)

The protocol will really depend on the properties of the protein that you want to isolate. Basically I use a lysis buffer like this:

KH2PO4 40mM

KCl 40mM

EDTA 2mM

Triton X-100 6%

Basic protocol

Mix cellular pellet with 4ml of lysis buffer. Briefly vortex and mix for 30 min at 4degC. Place solution in tubes and centrifuge at 30,000g for 60min. This will separate the lysed membrane from the proteins that are soluble in a 6% Triton soln. Check to see what your protein is soluble in. It may be that you need to alter the buffer.

I'm sorry that I can't add more to this protocol as the rest is specific to VDAC and is quite good at removing all other membrane proteins.

Well I hope that this helps you and I apologise for not having replied sooner.

1、因为我接下去是做western 的，所以只要膜蛋白在里面就可以了，这样是不是只要提取总的蛋白就够了，还是应该纯度高一些，提了总的蛋白之后再提取膜蛋白，这样会不会损失很大啊。但是你的方法好像是直接由细胞或者组织中提取膜蛋白是吗？那浆蛋白等在哪一步被分离了呢？我对蛋白的提取里面具体的原理过程不是很知道，但是觉得不知道原因，就这样照着做，很痛苦，谢谢你的帮助。

2、我目前的方法是：这是总蛋白的提取

液氮研磨组织，组织100倍量预冷的缓冲液A（10mmol/L Tris-HCL,320nmol/L 蔗糖，PH7.4，临用前加1mM的PMSF）；离心，每次10S×2，间隔20S，强度50，700g（4900rps），4度，10分钟。取上清，将此上清，37000g（18100rps），4度，40分钟，弃上清，取沉淀。用缓冲液B（A去掉蔗糖PH7.，临用前加1mM的PMSF）重新混悬。