The last step, what I have to do is that extract bacterial outer membrane proteins by ultracentrifugation. but I'm confused which speed is correct for the extraction.
I need only outer membrane proteins, not inner-, or periplasmic.
Some reference said, 20,000xg for 1 h won't be a problem for outer membrane proteins extraction.
But the other reference said, the condition should be at least 100,000xg for 1 h to extract outer membrane proteins.
Which speed is proper for the outer membrane proteins extraction? if neither of both, what speed is proper for that?
From your question it is difficult to know if you have enriched for an outer membrane fraction or not... but ultracentrifugation alone is not an acceptable method for the separation of bacterial outer membranes from inner membranes. Many researchers lyse the bacterium using a mechanical method (French Pressure Cell, Sonicator) and then separate the membrane vesicles (outer, inner, hybrid) using a density gradient for enrichment. Assuming that you have properly enriched for outer membrane vesicles by some method, the enrichment is typically ultracentrifuged to pellet the very small membrane vesicles at greater than 100,000 x g for 1 hour. I typically pellet membrane vesicles after enrichment by ultracentrifugation at about 150,000 x g for 1 hr.
Ultracentrifugation is not a method for extraction. It is a method for separation.Sucrose gradient ultracentrifugation is one method that can be used to separate outer and inner membrane prepared from mechanically disrupted cells based on the difference in buoyant density, as already discussed by James Carroll.
Another method that uses centrifugation to obtain outer membrane is to extract the total membrane preparation from disrupted cells, obtained by ultracentrifugation, with N-lauroylsarcosine detergent. This will dissolve the inner membrane but not the outer membrane. Then centrifugation is used to pellet the outer membrane. This can be done at a relatively low g-force, say 16,0000 g for 10 min, although the yield would probably be improved by using a longer time or higher g-force.
The outer membrane can be dissolved in n-octylglucoside detergent.
We have been using a version of the N-laurylsarcosine detergent extraction method mentioned by Adam Shapiro extensively. We do the centrifugation step more aggressively though (36,000g, 40 min), and it seems to generally work out well. You can find our protocol, along with the original references it is based on, in the following papers:
https://www.researchgate.net/publication/230789440_A_dual_tag_system_for_facilitated_detection_of_surface_expressed_proteins_in_Escherichia_coli
http://www.sciencedirect.com/science/article/pii/S0168165608001958
Article A dual tag system for facilitated detection of surface expre...
I think by the means of High Power Ultrasonic Waves
https://www.jstor.org/stable/30098301?seq=1#page_scan_tab_contents
Isolation of the Bacterial Cell Wall by Means of High Power Ultrasonic Waves
Giulio Bosco
The Journal of Infectious Diseases
Vol. 99, No. 3 (Nov. - Dec., 1956), pp. 270-274
📷JOURNAL ARTICLE
Isolation of the Bacterial Cell Wall by Means of High Power Ultrasonic Waves
Giulio BoscoThe Journal of Infectious Diseases
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