protein extraction - cytosolic lysis buffer (Apr/15/2008 )

Hi
i have a problem with my ripa lysis buffer, i added to about 1ml of the lysis buffer to 6cm dish for the extraction of the protein, but imediately the sample become very viscous and gummy and i dont know what to do?
here is the recipe of the RIPA buffer.

the protocol for RIPA buffer called for 137mM NaCl, 20mM Tris Cl, 10% glycerol, 1%Trixon-X-100, 0.5% sodium deoxycholate, 0.1% SDS 2mM EDTA


This is my stock solutions: 1.5 M NaCl, 1 Tris Cl, 50% glycerol, 10%Trixon-X-100,2%sodium deoxycholate, 0.1% SDS 0.5M EDTA

This is how much i added:9.1ul NaCl, 2ml Tris Cl, 20ml glycerol, 10mlTrixon-X-100, 25ml sodium deoxycholate, 10ml SDS and 400ml 2mM EDTA to make an 100ml of the RIPA buffer.

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It is probably goopy because of all the DNA and membranes. I'd try to clarify it by spinning it in a cold microfuge for 10 minutes.

What are you going to do with the lysate?
dan

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A bit of DNase always help remove the genomic DNA, which is most probably the cause of the viscosity.

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i was going to do western blot. I think i add to much of the SDS instead of Iml, i added 10ml

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thanks i will try that

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May I ask what is the speed u used to clarify the lysates? The lysates will be in eppendoft tubes for protein extraction right?

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the speed was 13000rpm

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hmm..13

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In my lab we have a very old centrifuge machine with temperature controlled. It is using the swing-out rotor. The maximum speed is 10000 rpm only. So can I spin my cell lysate at 10000rpm (it could be less than 10000 rpm becos the machine really very old and it can't reach the exact speed that we have set)? How long I need to spin the lysates so that I can get the protein in the supernatant?

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