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NCOR1 western


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#1 abrahas

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Posted 09 April 2013 - 10:01 AM

I am doing NCOR1 western on nuclear extract from Thp-1 cells in a gradient gel. Sometimes the band comes out beautiful, but the othertime across there is nothing significant above 250Kd marker. Because of that experiments have not been consistent. I used Lamin B as a loading ctrl which shows up always. Thanks

#2 bob1

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Posted 09 April 2013 - 12:37 PM

A few more details would be helpful - incubation time, incubation volume, washes, etc.

#3 abrahas

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Posted 11 April 2013 - 05:29 AM

ran it on a gradient gel. running buffer(50mM Tris, 380mM Glycine, 1.6mM EDTA, 0.1%SDS), Tranfer buffer(same buffer+10%methanol, w/o EDTA and SDS), block in 5%BSA/PBST, primary 1:1000 Rb anti-NCOR1(A301-146A, Bethel lab) in 1%BSA/PBST O/N at 4C, wash 3x 10min, secondary 1:2000 in 3%BSA/PBST, ECL Thanks

#4 bob1

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Posted 11 April 2013 - 12:46 PM

Assuming that this is the Nuclear receptor co-repressor protein (NCOR1) then my calculations put this at about 268 kDa. What was the gradient you ran?

How long and what voltage or amperage did you transfer at?

Try adding some SDS to the transfer buffer and removing the methanol.

#5 mdfenko

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Posted 12 April 2013 - 04:39 AM

you can use up to 0.05% sds in the transfer buffer, more may interfere with binding.

leave the methanol in the transfer buffer (you can go up to 20%). it will strip sds from the protein.

sds in the buffer facilitates the transfer of high molecular weight proteins but it must be stripped from the protein so that it won't interfere with binding.

see this handbook:

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#6 abrahas

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Posted 12 April 2013 - 04:54 AM

Initially I was running on a 5%gel then later switched to 4-20% when it was available. NCOR1 worked on both type of gels. It was run at RT at 100V which took about 1.5-2hrs, then transferred O/N(~18hrs) in 4C at 20V. I have added SDS in the past also but kept methanol at 10% in transfer buffer. The positive ctrl that worked couple of times does not work the third time along with my experimental samples. I like to move on but I just spend so much time on this to give it up.

#7 mdfenko

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Posted 12 April 2013 - 04:57 AM

maybe there's something wrong with your primary antibody?
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#8 bob1

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Posted 12 April 2013 - 02:28 PM

Or the samples are degrading from freeze/thaw cycles?

#9 mdfenko

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Posted 15 April 2013 - 04:14 AM

are you preparing fresh antibody solutions or reusing?

if reusing then you may have depleted the antibody.
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