CELL LYSIS - reasons behind protocols (Mar/20/2008 )
I am new in this forum and in the world of biochemistry as a whole. I am pretty confused by the protocols. I was wondering, is there any information online on the exact reasons why we use each reagent... for example... what is the difference between the tris-hcl and HEPES-KOH buffers? Why do we need two different trypes of buffers? I read somewhere that NaCl is used to agglomerate dna fractions. WHy do we need it in the first buffer when the nucleus membrane hasn't even been broken yet? My protocol doesn't even have sucrose.... which I hear is supposed to be important as an osmotic stabilizing agent. Please help.
What is your lysis buffer used for? DNA, RNA, protein, frationation...
Each type of buffer is designed to work for different things, for example, for making a protein lysate from whole cells I would want a buffer that breaks the cell membranes quickly and easily but not damaging the protein, so it would contain a detergent, a pH buffer (e.g. tris) and some salt so as to not damage the proteins on lysis and maybe not a whole lot else, whereas for a nuclear fractionation I would want a buffer that will gently lyse the cells and something that is iso-osmotic to the nucleus so it would probably just be a salt solution that is hyper-osmotic to the cytoplasm.
Tris is a pH buffer, and so is HEPES, they buffer different pH ranges though.
I suggest having a look at something like "current protocols" or "molecular cloning" by Sambrook et al., for your protocol and see what they have to say.
I see, I am doing cell fractionation in for the purpose of isolating and quantifying proteins in different cell fractions. My protocaol has two lysis buffers, the first one for the cytoplasm fraction and the latter one for the nuclear fraction. The ph seems to be the same in both buffers and I wonderd why the Nacl should be in the first buffer and not in the secind one when it is needed for the nuclear fraction.
2ml cold PBS with PI(Protease Inhibitor EDTA-) (3ml per 15ml dish)
Cell Lysis Buffer A 2ml
Stock Soln. Fraction
50 mM Tris-HCl(pH 7.4) 100mM 1ml
150 mM NaCl 2M 150µl
1% NP-40(Polyoxyethylene Ether)(mid-matsuda shelf) 100% 20µl
Protease Inhibitor (ETDA-)(needs to be x1 concentration) x10 200µl
Cell Lysis Buffer B 1ml
25 mM HEPES-KOH (pH7.4) 500mM 50µl
400 mM KCl 1M 400µl
1 mM EDTA 0.2M 5µl
1 mM DTT 100mM 10µl
Protease Inhibitor (EDTA-)(needs to be x1 concentration) 10x 100µl
Thanks for the explanation and the book recommendations. Ciao.