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Good afternoon,How can I decrease background in GST-pulldown...
Good afternoon,
How can I decrease background in GST-pulldown assay. I have been told I could use BSA.
At what stage of the protocol? How long, what concentration..?
In anticipation, thanks for your help,
Edith
(Brussels University)
How can I decrease background in GST-pulldown assay. I have been told I could use BSA.
At what stage of the protocol? How long, what concentration..?
In anticipation, thanks for your help,
Edith
(Brussels University)
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Lili Jing Author Answered Feb 28, 2013
Department of Cell and Molecular Biology, University of Pennsylvania, USA
Sorry for the belated response.
To reduce the background, you can try to use smaller amount of beads, shorten the incubation time and prolong the washing time.
You can also use BSA to pre-block the beads. For example, you can use 1-5% BSA (in your suitable buffer) to incubate beads at 4 degrees for a couple of hours to O/N. Then wash in buffer a couple of times before the addition of the protein lysates.
Hope it helps.
To reduce the background, you can try to use smaller amount of beads, shorten the incubation time and prolong the washing time.
You can also use BSA to pre-block the beads. For example, you can use 1-5% BSA (in your suitable buffer) to incubate beads at 4 degrees for a couple of hours to O/N. Then wash in buffer a couple of times before the addition of the protein lysates.
Hope it helps.
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Jian-Guo Zhang Answered Feb 9, 2013
The Walter and Eliza Hall Institute of Medical Research and The University of Melbourne
Edith: Not sure how strong your specific signal was in your GST-pulldown assay? If it was strong, in my opinion, the best way to reduce the non-specific background is to reduce the amount of Glutathione-Sepharose beads you were using in your experiment? A colleague you've never met.
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