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TAU gel electrophoresis of histones


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#1 pakbiochemist

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Posted 01 October 2012 - 11:40 AM

Dear Friends,

I have run 2D of my histones (1D=Triton acid urea gel electrophoresis; 2D SDSPAGE). Please comment on the results in the attached file. I am not getting it.

Regards
Binsan

Attached File  1.bmp   223.49KB   184 downloads

#2 pakbiochemist

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Posted 01 October 2012 - 11:43 AM

Dear Friends,


I have run 2D of my histones (1D=Triton acid urea gel electrophoresis; 2D SDSPAGE). Please comment on the results in the attached file. I am not getting it.
Attached File  1.bmp   223.49KB   140 downloads

Regards
Binsan

Please don't double post - bob.

#3 mdfenko

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Posted 02 October 2012 - 05:41 AM

have you determined how your sample runs in the first dimension alone?

you may not have sufficiently exchanged the buffer in the gel from the first dimension for the second or may not have sufficiently denatured the proteins after the first dimension (keep in mind that triton can displace sds).
talent does what it can
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#4 pakbiochemist

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Posted 03 October 2012 - 09:48 PM

in the first dimension they are poorly resolved. not very sharp and separated bands and the second dimension goes even worse.

What is mean by "not have sufficiently exchanged the buffer in the gel from the first dimension" I ddnt get this point

Regards
Binsan

#5 mdfenko

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Posted 05 October 2012 - 11:42 AM

if the result is poor in the first dimension it won't be any better in the second. you have to optimize the first dimension before going to the second. you should also optimize the second dimension technique before adding the first.

when preparing the first dimension gel slice for the second dimension you soak in a buffer that will denature the proteins (ie-sds/reducing agent). if you don't do this properly then your result will be variable.
talent does what it can
genius does what it must
i do what i get paid to do




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