I'm planning to separate proteins by size in total lysate(E. coli) by Amicon Ultra 0.5 - 30K, 50K, 100K (Millipore, UFC503008UFC505008UFC510008).
I heard total lysate would impair gel filtration column, so i tried Amicon ultra.
(The final purpose is checking wheter my target protein is soluble aggregates or not.)
I lysed E.coli cells by sonication in PBS, and used amicon ultra for protein separation by size. But almost all protein couldn't penetrate the membrane of amicon ultra, even though there are many proteins smaller than membrane pore size.
Is it impossible to seperate proteins in total lysate using Amicon ultra?
or Can i seperate proteins by size if i modify my protocol (such as add DNase, RNase, reducing agent, or some detergents in PBS buffer. or using mild lysis buffer rather than sonication.)
or are there any methods? please help me. Thanks.
Hello! I would guess that you are clogging the membrane with cell debris. If you wanted to survey the soluble fraction of your lysate, I would recommend some protease inhibitors (EDTA, PMSF, E64) and ultracentrifugation to remove the cellular debris before proceeding. As for aggregation, using your purification scheme (his-tag?) you could run a blue native page gel. BN-PAGE uses coomassie G for charge seperation and the proteins are not denatured. You would see multiple bands at an integer of the molecular weight of your protein of interest, if aggregating. If you dont have a purification scheme, I think that you could run a Western Blot afterwards. The Western may need optimization to get all of the proteins of interest separated. Also your antibody would have to work in ELISA because the proteins are not denatured fully by transfer to the membrane. Once you have your protein isolated you can check for aggregation with dynamic light scattering. If necessary you could then optimize your buffers to promote or resist aggregation. Good luck!
Oh. Thanks Matthew for your a lots of comments. I will try again with protease inhibitors. However, i have a question. I heard BN-PAGE first today, is it same with 2D-PAGE?
And actually, i had a plan to purify the protein with his-tag, but about 50% percent of protein was not purified, so i want to check the difference between purified target protein and unpurified target protein. Thus, i need a method for unpurified sample.
I think purified target would soluble and activie protein, and unpurified target would soluble aggregates or misfolded because their his-tag must be burried. I want to check this. Do you agree? or Do you have another assumption?
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