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brain tissue homogenization with sample buffer??


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#1 hanbecky

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Posted 19 June 2012 - 10:42 AM

Hi, I am trying to run a Western to detect a protein about 70kD in size.
We have tried traditional homogenization buffer (RIPA + protease inhibitors, ect.) or modified version of the buffers to homogenize the sample, but when we ran the gel, we didn't see the band on the target area but many many other bands in other places.

Then, our technician homogenized our sample directly with sample buffer (LDS Sample buffer from invitorgen) without any inhibitors and have a very nice band at the target size without any other unspecific band.

She said she heard some people homogenize the sample directly in the sample buffer, but I have never heard of that.
However, since she had a really nice band on the target size (no back ground what so ever.. very clean band), I am wondering if this method is also acceptable for the standard Western.

Dose anyone heard of it?
I am also wondering not adding protease inhibitor is OK for the sample's integrity.

I'll appricate for any suggestions or inputs.

#2 mdfenko

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Posted 19 June 2012 - 11:13 AM

the sample buffer should denature the proteases and phosphatases as well as the other proteins present in the homogenate. homogenizing samples in the sample buffer is done when the homogenate is not going to be used for anything else.
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#3 hanbecky

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Posted 19 June 2012 - 12:25 PM

Thank you!




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