I would like to isolate cell membrane proteins from human colorectal cancer cells (HCT-15) and human leukemia cells (HL-60). I am in search of a suitable protocol. I have to order all the materials and reagents for this experiment, as in our lab, we are doing this for the first time. So an easy protocol will be preferred. Please share you expertize with me. I will be grateful.
Best Regards!
If you want to fractionate the total membrane preparation into plasma membranes, ER, Golgi, mitochondria, and nuclei you can use sucrose gradient ultracentrifugation. Plasma membranes are the lightest.
You can use a differential centrifugation to separate nuclei and mitochonfrion. After that u need to use a ultra centrifuge to isolate the microsomal fraction, which needs to be solubalised in a buffer having a detergent.
Hi Salman! A good choice is to do, as Uddipan says, a differential centrifugaation. First, on my opinion, it is critical to carefully homogenate your cells by slight methods: I prefere srynge strokes (on a 23G insulin needle) for destroy cells without destroying other compartments. Then this homogenate you can centrifuge it at 245,000g during 90 min. Here you will obtain a cell membrane pellet. After that is very easy: if you want to release all membrane proteins, destroy the membranes by resuspending it with your choise buffer supplemented with detergents as CHAPS. For further info you can see the initial steps of this protocol in the paper of Reverter M et al, Molecular Biology of the cell 2011; 22 (21): 4108-23.
Regards
Juan
If you want to fractionate the total membrane preparation into plasma membranes, ER, Golgi, mitochondria, and nuclei you can use sucrose gradient ultracentrifugation. Plasma membranes are the lightest.
Hi, I have used this kit from life technologies for years, it used to be from Pierce:
https://www.lifetechnologies.com/order/catalog/product/89842
It works really well, you can see figures from my own work in my JBC paper from 2010.
Hi, If you have ultracentrifugation facility you can use sucrose gradient as suggested. Only disadvantage of this method, it requires large amount of cells since the yield is not so high. As an alternative method, you can use biotin labeling and pull down your surface proteins with streptavidin beads. This method is quite easy and works well, you can see the protocol and WB images from the paper below.
http://www.ncbi.nlm.nih.gov/pubmed/25476450
Bests
See: Article Strategies for the Purification of Membrane Proteins
orArticle Chapter 35 Purification of Membrane Proteins
orArticle The Use of Detergents to Purify Membrane Proteins
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