Because of a purpose of my project, I have to lyse cells in low pH. What is the lowest pH for cell lysis? Lower is better for me. Protein denaturation is not big deal, even cleavage of disulfide bond is okey.
Additionally, the component of the lysis buffer which I use is 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 50 mM Tris-HCl (pH 7.5), EDTA (pH 8.0).
Have you tested out this buffer on cells before? Honestly, your best strategy is to just try it on a few extra samples and see what kind of yield you get.
Low pH is actually very helpful for cell lysis. You do not need Triton and SDS. Boiling and pH 3 is a simple way of achieving lysis, if your protein can take high temperatures. You can go down to 70-80 degrees and still achieve lysis.
good luck
@ Katie A Clark
I tried that with the above lysis buffer with pH 3.0, but it shows cloudy and turbid solution. I suspect that the pH is too low to induce aggregation of proteins.
@ Leif Bülow
Thanks for your kind answering. I'd like to avoid the boiling step before pull-down step. Is there any good alternative?
I often use a lysis buffer with pH 8 to lyse bacterial cells after freeze-thawing cell pellets. It works well for me. You can also use sonication for efficient lysis. The important point here is the stability of your desired protein in lysis buffer.
Of course, you'd not use Tris to buffer below 7 or so. Phosphate, Gly or Ala can buffer around pH 2, this is probably the lowest pH at which buffering makes sense in aqueous media.
EDTA is poorly soluble in acidic media, it also looses its ability to complex many metal ions, in particular those of group II of the periodc system.
SDS is poorly soluble in acidic solutions. Up to a point, you could use LDS (lithium dodecylsulphate), but you may be better off replacing the anionic detergent by a cationic one like CTAB. Contrary to what some books claim I have found CTAB very benign towards proteins (I have not tested it with nucleic acids). You can even perform cationic electrophoresis in CTAB (doi:10.1016/S0003-2697(02)00639-5) with those samples, thereby avoiding buffer and detergent exchange.
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