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lysate buffer


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#1 wuxx0153

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Posted 25 February 2009 - 09:42 AM

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. :huh: :(
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.

#2 bob1

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Posted 25 February 2009 - 03:58 PM

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.)

#3 Nrelo

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Posted 01 March 2009 - 08:06 PM

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

#4 wuxx0153

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Posted 02 March 2009 - 09:15 AM

Thanks for informations, it help a lot.

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

#5 bob1

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Posted 02 March 2009 - 03:24 PM

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

#6 Nrelo

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Posted 04 March 2009 - 03:55 AM

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

#7 madrius1

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Posted 04 March 2009 - 07:37 AM

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).




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