I am expressing a protein that should go to the outer membrane of E.coli and I would like to visualize my expression using SDS-PAGE. I have tried running whole cultures and bug buster+nuclease lysed cultures. These were also reduced using 1x standard protein loading buffer containing and loaded onto the gel. I get many bands from the cell lysates but not the required band at ~23kDa. Is it probably because outer membrane proteins are hydrophobic and do not get solublized easily? Is there a better way to do this?
if i understood your expirment conditions right, i do think that your problrm lies in the buffer u r using, i advice you to lysate your cells using the NP-40 or RIPA buffers. and i do strongly recommend RIPA in case you are not sure your protein in completely extracted using you ordinary buffer
You must prepare an overnight culture of your bacteria and then add sample buffer to it (1 sample/ 3 buffer) and lets it is boiling for 15 min in 100c bath. finally you must do SDS-PAGE by 12% gel for 2.5 hours by 32 mA. You can use ladder that detects between 11-250 KD.
Do not sonicate your samples because it cause secretory proteins get release.
do a mechanical shearing using a syringe with a fine bore like insulin syringe. try the lab protocol or increase the percentage of detergent like NP-40 or tween or triton in your lysis buffer. I wwould suggest NP40 if the protein is sensitive but you have to play with your conditions...
We used the bugbuster master mix to get membrane protein too. The expression protein is in the pellet after centrifugation. Then we purified the membrane protein by the method of inclusion body purification according the Novagen prtocol. After all the protein can be deteced on the SDS PAGE.
Outer membrane proteins are generally perfectly solubilised in buffers containing SDS (assuming your using that in your buffers). However, membrane proteins (in particular beta-barrel proteins) tend not to unfold completely in SDS and/or bind more SDS than an average water-soluble protein. Both effects can contribute to a significant change in electrophoretic mobility; in other words, your protein might run at much lower or higher molecular weight than expected. You could try running a Western blot to determine whether your protein of interest is expressed and at what molecular weight it runs.
I agree with Guus.
Also, study what others have published regarding how to handle outer membrane proteins. If the papers lack sufficient experimental details write a letter of complaint to the editor, and/or find other papers....
Standard procedure for membrane proteins is first a centrifugal isolation of membrane fragments after lysis (=getting a "membrane preparation" - done to enrich the protein of interest (POI) into a smaller volume, while also getting rid of soluble cytoplasmic proteins in the supernatant). Without such an enrichment, the POI may be impossible to detect - if so, trying to solubilize directly in the lysate will fail.
Then follows solubilization of the membrane preparation with a suitable detergent. That detergent should have a good solubilization efficiency for the POI, while at the same time preserve the POI activity (if you are interested in the activity). Choice of detergent is often a trial-and-error approach, with screening of several detergents.
For detection purposes only (SDS-PAGE and Western), solubilization with the very strong and denaturing detergent SDS can be used.
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