Hello everybody!
Could somebody explain me more about the effect of detergent in the wash buffer of a co-IP?
Dear Donna Schweizer
it is common to observe the positive control in the unbound fraction/flow through. The binding capacity of GTMA M-270 is 2-2.8 µg of recombinant GFP per 25 µL bead slurry which is lower than standard GTMA beads; however, practically it even binds to lower amount of GFP-tagged protein (depending on the size, type of the protein). Check if your sample is oversaturated with the protein of interest (for instance, if your protein of interest is overexpressed).
The use of concentration of detergent in wash buffer is to wash out the non-specifically bound protein from the protein complex. The more important task is to check if the upstream steps of your co-IP is accurate. Check if the beads are equilibrated in appropriate buffer solution. Do not incubate for too long, but incubate in cold temperature in a rotating mode. Check the wash buffer compatibility table if you overload any of these reagents. Do not vortex at all when the beads are added, and treat them gently (by pipetting up and down for a few times and magnetic separation). If you want to isolate a bulky native complex of protein, then you may ignore detergent in washing steps, but then you need to worry about non-specific binding.
All the best!
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining