For isolation of membrane proteins (inner or outer membrane) the best is to use a French press to break the cells. In principle adding lysozyme, DNAse, RNAse is not neccesary to lyse the cells with a French press. After lysis do a short spin (15 min) at a low speed to get rid of the cell debris.

After this step there are two options. If you want to get a very pure outer membrane fraction you have to do a sucrose gradient. For this you first have to collect all the membranes (= 1-1/2 hours at 135,000 g). The membrane pellet (= IM + OM) you take up in a very small volume and subsequently you load this on a sucrose gradient whereafter you centrifugate again. (this takes +/- 12 hr). After centrifugation you will have four bands in the tube. The upper two are the inner membranes (yellow brown), the lower (whitish for E.coli), the outer membranes.

For less pure outer membranes you can use the following "trick".  After you have removed the cell debris. Put the solution in an ultracentrifuge tube, put it in the ultracentrifuge, wait till the vacuum has installed and start the centrifuge. Once the centrifuge has reached the 135,000 g wait 1 minute and then stop centrifugation. The membrane pellet that you obtain with this protocol is mostly the outer membrane. (By centrifugation the rest of the solution for 11/2 hr at 135,000 g you obtain a membrane fraction enriched in inner membranes).

Outer membrane proteins are often very difficult to extract. SDS works perfect but your protein will be for sure denatured. So if you want to do functional studies afterwards you have to find a detergent or conditions that are capable to extract the protein(s) of your interest  (non-ionic detergents in general do not work for outer membrane proteins). My best experience for extraction OM proteins were zwitter-ionic detergents like SB3-12 and SB3-14.

Dear Jeongjin , I see too many steps to look for proteins in the outer membrane. Are you following a specific protocol for isolation of outer membranes. In my mind this would be the first step (isolation of outer membrane with detection of appropriate markers) and after that to extract the proteins in this membrane.

Going to your questions

Protocol is key

Protease: any commercial cocktail should be OK

Sonication (mild) do not damage proteins but messes up all the contents and then you cannot say what comes from where. 

Storage at 4, depends for what and for how long.

Proteomics: difficult to recommend as you would first decide which is your main objective.

I assume that you are not interested in periplasmic but on proteins integrated within the membrane components. 

Best

The 10-minute sonication is a bit of a worry, since it could result in overheating of the sample unless plenty of time is allowed for cooling between bursts. My preference would be to use a French press to break the cells. A bead beater is another option, although sample heating is also an issue with that device..

People often include Mg2+, lysozyme, DNase and RNase during the homogenization step in order to break down the peptidoglycan, DNA, and RNA. Breaking down the DNA is particularly helpful in reducing the viscosity of the sample.

Your 20,000 g centrifugation will pellet both inner and outer membranes, although perhaps not quantitatively. A 100,000 g spin for 1 hour will improve the yield.

To get a purer preparation of outer membranes, you can extract the pellet with 1% N-lauroylsarcosine, which will dissolve the inner membranes but not the outer membranes. Then you can centrifuge again to collect the outer membranes.

For isolation of membrane proteins (inner or outer membrane) the best is to use a French press to break the cells. In principle adding lysozyme, DNAse, RNAse is not neccesary to lyse the cells with a French press. After lysis do a short spin (15 min) at a low speed to get rid of the cell debris.

After this step there are two options. If you want to get a very pure outer membrane fraction you have to do a sucrose gradient. For this you first have to collect all the membranes (= 1-1/2 hours at 135,000 g). The membrane pellet (= IM + OM) you take up in a very small volume and subsequently you load this on a sucrose gradient whereafter you centrifugate again. (this takes +/- 12 hr). After centrifugation you will have four bands in the tube. The upper two are the inner membranes (yellow brown), the lower (whitish for E.coli), the outer membranes.

For less pure outer membranes you can use the following "trick".  After you have removed the cell debris. Put the solution in an ultracentrifuge tube, put it in the ultracentrifuge, wait till the vacuum has installed and start the centrifuge. Once the centrifuge has reached the 135,000 g wait 1 minute and then stop centrifugation. The membrane pellet that you obtain with this protocol is mostly the outer membrane. (By centrifugation the rest of the solution for 11/2 hr at 135,000 g you obtain a membrane fraction enriched in inner membranes).

Outer membrane proteins are often very difficult to extract. SDS works perfect but your protein will be for sure denatured. So if you want to do functional studies afterwards you have to find a detergent or conditions that are capable to extract the protein(s) of your interest  (non-ionic detergents in general do not work for outer membrane proteins). My best experience for extraction OM proteins were zwitter-ionic detergents like SB3-12 and SB3-14.

From past RG inquries:

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=6&cad=rja&uact=8&ved=0ahUKEwjrls7pp4jNAhVO7WMKHeDhDD0QFghHMAU&url=http%3A%2F%2Fwww.sciencedirect.com%2Fscience%2Farticle%2Fpii%2FS0944501314001153&usg=AFQjCNHkIYSJ9lNjcjHm80UE_9cInJ6CXg&sig2=W9RyvG3LBOTKAYr4j7NB4w

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=4&cad=rja&uact=8&ved=0ahUKEwjrls7pp4jNAhVO7WMKHeDhDD0QFgg4MAM&url=http%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC2763183%2F&usg=AFQjCNFYaHQvIcx7CyQMQ-N7XGRpjZRVqA&sig2=30esWx8CIZf5XPuHpl4TUQ