4 replies to this topic

[pakbiochemist]

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

 1.bmp   223.49K   76 downloads

[pakbiochemist]

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.
 1.bmp   223.49K   64 downloads

Regards
Binsan

Please don't double post - bob.

[mdfenko]

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).

[pakbiochemist]

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

[mdfenko]

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.