I need to isolate the membrane proteins to evaluate the effect of a treatment on this cell fraction.
Purification of membrane protein is still a difficult issue in Biochemistry.
I have previously purified lipoamidase from pig-brain membranes by using NP-40 (please see file; Purify Brain Lipoamidase). Both Triton X-114 and NP-40 (c.a. octylated surfactants) are not sufficient to dissolve the human bile (please see file; HPLC-Surf-SEC protein determination method).
I am now thinking that in order to improve the recovery of membrane protein from tissues, we should use more hydrophobic non-ionic detergent of Brij-58 (palmitoylated surfactant). Brij-58 is good for separating membrane proteins and fucoidan (please see files; SEC column 300 A silica and JCB Fucoidan transport).
It seems that the narrower distribution in Mr of surfactant is very important. The Brij-58 of Sigma seems to have wider Mr distribution than Wako's, and the use of Wako's surfactant has only given a good result in fucoidan determination (our unpublished observation).
Purification of membrane protein is still a difficult issue in Biochemistry.
I have previously purified lipoamidase from pig-brain membranes by using NP-40 (please see file; Purify Brain Lipoamidase). Both Triton X-114 and NP-40 (c.a. octylated surfactants) are not sufficient to dissolve the human bile (please see file; HPLC-Surf-SEC protein determination method).
I am now thinking that in order to improve the recovery of membrane protein from tissues, we should use more hydrophobic non-ionic detergent of Brij-58 (palmitoylated surfactant). Brij-58 is good for separating membrane proteins and fucoidan (please see files; SEC column 300 A silica and JCB Fucoidan transport).
It seems that the narrower distribution in Mr of surfactant is very important. The Brij-58 of Sigma seems to have wider Mr distribution than Wako's, and the use of Wako's surfactant has only given a good result in fucoidan determination (our unpublished observation).
Dear Marcel,
If you need a "fast" protocol to test your samples you can lysate your cells in ice cold hypotonic buffer to break the cells without solubilize the membranes (there are several recipes available on line). Load the lysate in a 1M sucrose cushion and spin at 100000 xg in ultracentrifuge for 1 hour. You'll end up with 2 phases and a pellet. Upper phase is mostly cytosolic content, membranes remains in the interphase and the pellet at the bottom is enriched in cytoskeleton and nuclear components. It can be considered a fast and dirty protocol but I think is quite efficient, specially for pilot tests.
Best,
Thank Dr Dupraz, I will run a pilot study using this "fast" protocol !
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins