I am doing protein mapping to my interesting protein (chop it off for different domains, mix them with other GST- protein to do in vitro binding assay and then they are pulled down with GST- beads), but I realized that some domains bound to GST-beads in the controls. Is there any suggestion?
Agree with Paul. Also try pre mixing your beads with 0.1% BSA in your binding media to block non-specific sites.
Hi, first, thanks for your kind suggestions.I used 0.1% Triton x-100, but did not work. Also, increasing the salt concentration, inhibit the binding between domain(s) and GST- protein. I am trying to block the beads with 5% milk and will see!. Please, Mr. Bardbury could you provide me more details about the blocking with BSA specially blocking time? do I have to wash the beads after the blocking ( before they are added to the protein mixture)? Are there any significant differences between blocking by 5% milk and 0.1% BSA in my case?
Thanks
Wash beads 3X with PBS with 0.1% BSA then incubate overnight at 4'C, then wash beads 3 times with PBS. Hope this clears up your binding.
I have been trying pull down experiments with GST tagged proteins an have had the same problems. I tried buffers with high salt and detergent and blocking with BSA as suggested above. I also tried reducing the incubation time. It might also be worth checking that your proteins are not binding the glutathione sep- beads. And if they are try an in solution assay (without pulling out with the beads). Hope you manage to sort it out.
Thanks for all. Mrs. Sadler, could you give me more details about solution assay and how I can do it?
I appreciate your time
Regards,
you cannot.... you will be able to minimise it somewhat but u would be amazed if you got rid of it , i would do the best you can with varying salt concentration and detergent, maybe try changing buffering (Hepes instead of tris for example) then when you have the prep as good as you can get it i remove the GST (if that is desirable) then use a size exclusion column to further purify the protein of interest
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