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applying cell culture protocol of protein extraction for rat intact tissue is co


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#1 pakbiochemist

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Posted 19 October 2011 - 11:37 AM

Dear all,

I am following a protocol which is based on histone extraction from cell culture and i am applying it on my rat brain tissues. I have 2 questions

i. What weight of tissue is equivalent to 2.5 x 106 cells?
ii. The protocol uses lysis buffer(MgCl2, KCL, Tris, DTT, PMSF, protease inhibitor, phosphatase inhibitor) without homogenization. only incubation with lysis buffer for 1hr. is it possible for the cells to lyse without homoganization in lysis buffer or shud i incorporate a homogenization step

Regards
Binsan

#2 bob1

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Posted 19 October 2011 - 04:21 PM

As you have a mass of cells, you will need to homogenise the tissue. For tissue culture cells, they are typically resuspended in the lysis buffer.

The mass of tissue will vary with tissue type for that number of cells - brain cells are mostly rather large and quite fatty (less dense) so you will probably need a quite a bit. I would start with about 100 mg.




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