Hi Sepideh,

Would you please explain more about the process of your work? I’m not sure which step do you mean! Extraction of recombinant protein from periplasm or purification?

Dear Sepideh Parsapour

thaeoretically if your protocoll work well, no cell lisys have to happen during the sucrose incubation, while the rapid passage from high sucrose/no sucrose (with conseguent shock in terms of osmotic pressure) will induce periplasm lysis ans protein release from surnatant, so if all work well (you can check it by loading n SDS page the different fractions), the periplasmic protein are contained in the mgs04 solution and you do not need to dialize it but you can simply dilute in binding buffer for IMAC and load in into the coloumn to perform protein purification.

best regards

Manuele

Dear Sepideh Parsapour

thaeoretically if your protocoll work well, no cell lisys have to happen during the sucrose incubation, while the rapid passage from high sucrose/no sucrose (with conseguent shock in terms of osmotic pressure) will induce periplasm lysis ans protein release from surnatant, so if all work well (you can check it by loading n SDS page the different fractions), the periplasmic protein are contained in the mgs04 solution and you do not need to dialize it but you can simply dilute in binding buffer for IMAC and load in into the coloumn to perform protein purification.

best regards

Manuele

Hi Manuele Martinelli

I’m so thankful for your answer. I have just a few questions. Could you please explain your procedure in more details?

Maybe It is a funny and simple question, but should I prepare MgSO4 buffer in water or in the previous buffer (30 mM Tris pH8)?

Dear Sepideh Parsapour

personally i never used the mgcl2 in the buffer. I used a protocoll simila to the ohe reported for E.coli on:

http://cmdr.ubc.ca/bobh/method/osmotic-shock-procedures/

the protocoll suggest to use water, probably to maximize the shock but it depends if your protein is stable on water or it need at least a buffer to obtain a pH far from its pI.

in some case (eg fos Cu, Zn SOD1) water worked well for me, in other cases, with less stable proteins, i used tris 20mM pH=8 and it is works well too.

I tihnk that you can try to do some test in small scale with water and some buffer and see in SDS-page which is the best condition for your protein.

best regards

Manuele

Dear Atousa Aliahmadi

Thank you for answering my question. I have problem in extraction of recombinant protein. First, we resuspend cell pellet in sucrose buffer (30mM Tris, 20% sucrose, 1mM EDTA, pH8).then after incubation on ice for 30 min and centrifuge the cell suspension. We just keep the supernatant from sucrose buffer as S1 on ice. After that resuspend the pellet from previous centrifuge in MgSO4 buffer and incubate for 10 min after that we add EDTA dropwise to final concentration 1mM.after incubation on ice for 5 min, centrifuge the cell suspension. In this step after centrifuge we collect supernatant as S2. In final step we mix 2 collection supernatant (S1&S2) and then dialysis these mixture with pbs buffer overnight.

my question is am I doing the right method? I mean mix these two supernatant(S1&S2)?

for prepare MgSO4 buffer should i use water or Tris buffer pH8?