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Lysis Buffer for Total Protein Assay


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#1 Lupo Comunitario

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Posted 08 April 2009 - 04:54 AM

Dear All

I need to measure the total protein concentration from some P.fluorescens (GRAM -) cultures and I would like to use the Bradford assay.

I would lyse the cells by sonication or bead beating. Could anybody suggest me which lysis buffer to use?
Could it be directly used for the Bradford assay or should purify the proteins further?

Thanks a lot

#2 Dr Teeth

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Posted 08 April 2009 - 05:25 AM

Use a RIPA buffer with sonication (look up a recipe online, typically: 50mM Tris-HCl pH 7.4, 150mM NaCl, 1-5% NP40, 0.25% Na-deoxycholate, 1mM PMSF).

You can then use this lysate directly for a Bradford assay if you remember to add a volume of buffer to your blank that is equal to the volume of sample you are adding to your protein samples as NP-40 alters a coomassie reading slightly.

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley




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