Abstract
GST-Pull down assay is an effective way to examine the direct binding of two proteins in vitro. This protocol is based on GST pull down system from GE healthcare, and uses the binding of unplugged/MuSK receptor and Wnt ligand as an example to illustrate the detailed procedure.
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was developed in the Michael Granato Lab at University of Pennsylvania, Philadelphia, USA, and this work was supported by NIH grant R01HD037975.
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
It should be GST beads.
YES.
Thank you!
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.
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.