I get multiple protein bands in SDS PAGE after expression in BL21 cells. Does sonication results in protein degradation and result in extra overexpressed bands?
I sonicate for 30 seconds with a gap of 15 seconds at 60% amplitude for 5 cycles on ice. I try to get a clear solution during sonication.
Can anybody give me the right explanation?
Thanks.
Hi Gaurav,
Your question is not clear. You getting the multiple bands after purification or you talking about SDS PAGE gel of whole supernatent fraction or whole cell lysate?? if you clarify this we can suggest you some explanation.
Sometimes you have overexpression of endogenous proteins of E. Coli such as chaperonines or just the drug resistance protein when you are overexpressing an exogenous protein. This may be due to that the protein that you are expressing is harmful for the cell, so the bacteria overexpresses these proteins which help to reduce the damage.
If you want to avoid those overexpression bands, you could produce your protein in lower temperatures (such as 16 or 18 ºC), that will help a little bit.
Hi there,
If you want to know when this extra bads form, compare samples before induction, after induction and after cell lysis. For the first two samples, boil harvested cells directly in SDS/PAGE loading buffer. If the bands appear only in the lysate then it has to do with extraction condition.
@ Gaurav Chhetri- I have written about cell lysate supernatant fraction and not purified protein or whole cell lysate. My construct is soluble one and it is being expressed in pET28a vector with a His Tag at C-terminal.
@ Dominique Liger- Your suggestion looks logical. I will try to apply your suggestion. I should get same protein bands in the boiled samples as in case of cell lysate supernatant fraction if the sonication is going well.
@ Francisco Rivero- I am doing expression at 16 degree overnight in rosetta cells. I am expressing a beta-lactamase protein.
Thanks..
Well, if you are already expressing at low temperatures i think you can not get rid of those other bands (Unless if you try with other E.coli Strain), but maybe afterwards, when you purificate the protein (if you intend to), you might get the protein pure.
Which method are you going to use to purify? (if so)
Did you add protease inhibitor during sonication? If not you can try to purify it again adding protease inhibitor. Another thing is high amplitude also can degrade protein. In my case, I use 40% amplitude. You also can try this.
Good luck
Try adding some EDTA-free protease inhibitor tablets with some benzamidine and PMSF in the buffer you resuspend you cells in before sonication. I had a similar problems and this massively reduced the extra bands I was seeing.
Gaurav,
I am totally agree with Mr. Francisco Rivero that sometimes you have overexpression of endogenous proteins of E. Coli. You should try to purify your protein and check it with ant His antibody. If your protein is there you shouldn't worry about other expressed proteins.
Dominique is correct you need to go back to basics and run a gel. Do a growth curve and run a gel with uninduced, the induced after 1, 2, 3, 4 hours etc. Try to normalise loading using the OD600 of the growth and remember after an OD of 1 dilute 10 fold to get a more linear reading. Then run everything on the gel possibly with your soluble and insoluble fractions as well. Often certain bands get concentrated in the soluble fraction. If you see multiple bandson induction it could indicate that you have problems with toxicity and you are getting deletions- I have had this problem in the past with a pathogenicity protein.
In addition to the above suggestions, and in case you see degradation during sonication, that may be due to protease activity, which can easily be controlled by protease inhibitor cocktails, in most cases. Roche has some nice tablets that I have used in the past, just beware of EDTA, if you are going to purify the protein with metal binding columns.
Thanks alot to all of you.
I have still a big query. I am using protease negative expression host (i.e. BL21 and Rosetta E.coli strains). Why should I use protease inhibitor?
@ Francisco Rivero- I am trying to purify a beta-lactamase protein which has His-Tag at c-terminal.
Hi!
As Francisco Rivero and the others said try the useful tips, but if your protein has a his-tag don't worry. You should have a quite pure protein after the hi-trap column. Protease inhibitors can help you, because protein degradation can reduce your pure protein yield.
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