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membrane protein purification


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6 replies to this topic

#1 jasmina

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Posted 16 February 2010 - 01:03 PM

Hi,
I'm trying to purify one membrane protein, which is solubilized in RIPA buffer containing 1% triton x-100, Na DOC, Nacl, EDTA, SDS and Mgcl2.
The problem is when I try to quantify the samples by bradford, the cuvettes are with intense blue!!! even the cuvettes of BSA for the standard courb.
I'm wondering which compound of RIPA that interfere with bredford!! I tried also by turbidometric method TCA; all the samples are precipitated before the quantification by spectrofotometer.
Please, if you have any suggestions,
thanks
waiting for reply soon

#2 fishdoc

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Posted 16 February 2010 - 03:35 PM

Bradford is not compatible with high concentration Triton X-100 or deoxycholate.

#3 jasmina

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Posted 16 February 2010 - 03:41 PM

thanks for your reply.
I will try today to change this parameter.

#4 jasmina

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Posted 16 February 2010 - 03:54 PM

For RIPA buffer, i read that bradford is convenient. should I try TCA or keep RIPA and replace triton by another detergent and quantify by bradford?
In fact, I read in the literature some protocols using RIPA with the same components as mine, but, i don't know how they quantified proteins!!
thanks for advice

#5 mdfenko

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Posted 17 February 2010 - 08:02 AM

you can download the protein assay technical handbook from thermo-pierce to help guide you in selecting an appropriate assay.
talent does what it can
genius does what it must
i do what i get paid to do

#6 jasmina

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Posted 17 February 2010 - 08:59 AM

thanks alot

#7 jasmina

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Posted 17 February 2010 - 03:34 PM

I tried to dilute my ripa buffer. as a trial, i solubilise one sample on diluted ripa, and color was a bit good ( not blue like non diluted ripa!).
I'm thinking to use the same ripa buffer diluted since I read it also in the "Thermo scientific total protein assays", dilution 1:10. without changing triton 1% or deoxycholate, I will see if it works, to quantify my samples!!!
thanks for interaction




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