Protein expression in e.coli - (Jan/23/2013 )
For checking the protein expression in e.coli, till now in our lab we've been doing sonication (for miniscale 5ml culture) , separating out the supernatant and pellet and loading them onto gel. That tells if the protein is present in pellet or in supernatant. But I have seen people using some quick method where you just add some 1-2% SDS or gel loading dye in culture pellet (before sonication), heat it and load on the gel. I am not aware of this. please guide me. How SDS is just enough to lyse the cells, then why do we do sonication?
SDS/protein loading dye impact negative charges and linearize proteins. I prefer to sonicate and add protein loading dye after (SDS can become frothy and escape tube). Either way will work though.
jerryshelly1 on Thu Jan 24 01:34:32 2013 said:
Either way will work though.
My question is, sonication is done for lysis of cells. E.coli is having cell wall, so only addition of SDS is enough to lyse the cell? How can we see protein by just adding SDS and heating it? How does it work?
Sorry, I misunderstood the question. I think you may be referring to colony cracking. I have never seen it done with SDS by itself, but I have seen NaOH added to lyse the cell. You should look up alkaline lysis and colony cracking. Links are below.
I hope this helps
After a quick Google search for "SDS bacterial lysis, wikipedia has some answers
By using SDS loading buffer and heat you get total protein but you don't know whether your protein is soluble or not (crude total lysate is very dirty for downstream purification processes, and specially gloppy due to DNA). Is a good quick method to test whether your protein is being expressed at all. When I was doing loads of protein expression, a few years ago, I always used this method. I used to take samples at different time points (from T=0 no expression), then just mix with loading buffer, heat, spin down and load.
and here's the Wiki link, I've copied the part on detergents.
@ Jerryshelly1,Thanks for the links. But again you misunderstood , it was not about miniprep. It was for protein. next post by almost a doctor explains what i was talking about. But thanks for the effort
@Almost a doctor, Thanks, You got it right, this is what I wanted to know. This method is quick, why dint I know this . I will try this now, just add SDS dye in the pellet, heat, spin and load, right? Is there specific % of sds that we need to maintain?