6 replies to this topic

[wuxx0153]

I am asked to modify our protein lysate buffer from cell lysate to tissue lysate, but also need to keep some protein enzyme activity.

I just realized how little I knew about the function of the components in the buffer.     
Can anyone suggest any reference(s) (book is fine, but web site will be great) that summarize the function of each component and purpose of using them in buffer.

[bob1]

I don't have any references, but lysis buffers boil down to three things:

pH buffer (tris, hepes, mops etc.)
Salt concentration/osmolarity (NaCl, KCl, MgCl₂, CaCl₂ etc.) - usually aiming for a hypo-osmotic solution.
Detergent (SDS, Triton, Tween, Deoxycholate etc.)

[Nrelo]

For lysing cell lines, include 1% Triton X-100 into the lysis buffer,this detergent will solubilize cell membrane and has little effect to protein integrity.

Typical cell lysis buffer contains:
50~100mM Tris
150mM NaCl
1% Triton X-100 or NP-40
Protease inhibitors (EDTA, PMSF etc) or you can use commercial protease inhibitor cocktail

[wuxx0153]

Thanks for informations, it help a lot.

as I understand, tissues are tougher for regulare lysate buffer.
So which component should I increase?

[bob1]

detergent:  it dissolves the cellular membranes and increases solubility of proteins.

[Nrelo]

wuxx0153, on Mar 2 2009, 09:15 AM, said:

Thanks for informations, it help a lot.

as I understand, tissues are tougher for regulare lysate buffer.
So which component should I increase?

in that case you need to first digest the junction between cells to obtain cell suspension. i have no epxerience on this, you might ask those experts in the cell biology area

[madrius1]

Increasing some component could result in protein damage/degradation. The optimization of the method should be done in the homogenization part, where you reduce the physical integrity of the tissue to obtain a "suspension". This can be done by etheir collagenase digestion or mechanical ways (powdering the tissue in liquid nitrogen, grind with beads, polytron, sonication, etc).