2d gel cleanup

David J. Meyer, Ph.D. meyerdj at phibred.com
Wed May 31 09:55:57 EST 2000


You most certainly can use TCA-precipitated proteins for 2-D gels, with
excellent results. Care must be taken to remove the TCA before you run the
gel, though!

TCA would coprecipitate the charged (radioactive) tRNAs, so they would not
be removed.

David J. Meyer, Ph.D.

"Hiranya S. Roychowdhury" wrote in message
<200005192104.PAA259858 at nestor.NMSU.Edu>...
>You can not use TCA precipitated proteins for 2-D gels.
>It is also hard to imagine how you would have 35S signal all over the gel.
I
>think you are using too much excess of the labelled amino acid. Even then
>you should not see the labelled aa "all over the gel"
>
>However, you can use sephadexG25 spin columns to clean up the crude protein
>extract following the labeling.
>
>At 01:49 PM 5/19/00 -0500, Andrea Beckel-Mitchener wrote:
>>I am new at 2D gel electrophoresis and would appreciate some technical
>>advice.
>>
>>I have synthesized proteins (labeled them with 35S-Met) and run a 2D
>>gel.  There is quite a bit of background from the radioisotope, yet I am
>>able to see very good "spots" as well.  I would like to clean up the
>>appearence of the gel by possibly getting rid of unincorporated label if
>>possible  (much of the signal runs with the dye front and doesn't pose a
>>problem, it's the rest of it that is annoying).  Can I precipitate
>>proteins and still run a good 2D?  I was concerned that precipitation
>>with TCA might interfere with running the IEF dimension.  Any
>>suggestions/advice would be great.  Thanks.
>>
>>--
>>Andrea Beckel-Mitchener, PhD
>>Beckman Institute
>>University of Illinois
>>
>>
>>
>
>
>Dr. Hiranya Sankar Roychowdhury
>GENE LAB/ EPPWS
>New Mexico State University
>Las Cruces, NM 88003
>Ph. (505) 646-5785
>hroychow at nmsu.edu
>
>---
>







More information about the Methods mailing list