I want to identify endogenous binding partners for a protein I'm studying. I was planning on making a protein extract from the tissue I'm interested in, incubate this protein extract with the affinity purified antibody I've made against my protein of interest, then purify with Protein A-Beads and do Mass spectrometry on the pulled down material. I have never done this before, and all the related protocols I've seen online involve tagged proteins. The TAP method seems that would be the best for my purpose, but there's no LAP-tag transgenic mouse line available for the specific protein I'm working on, and it would be too much of an investment for us.
Any suggestions or protocol about affinity purification of protein complexes from in vivo tissue extracts would be very welcome.
We use a BioRAd sequential extraction kit that separates proteins into 3 extractions based on hydrophobicity.
I have seen papers where Sephacryl gel filtration or ultracentrifugation in glycerol gradients were used. Gel filtration media are available for HPLC but the columns are very expensive. Something you need to consider is a predicted molecular weight and (perhaps) molecular shape. Also you should consider how you plan to maintain the complex in vitro (ions, pH, ATP, ADP or other small molecules).
Thanks for all the answers. I see that I'll have to go through some quite a bit of testing!
- Badges
- Science topic
- Similar topics
- Papers