docs/Protocols/Gentle coIP protocol.md

name, id, version, description, author, created, materials, steps, notes

name	id	version	description	author	created	materials	steps	notes
Gentle Co-Immunoprecipitation Protocol	PROT-0012	1.0	A gentle co-immunoprecipitation protocol for preserving protein-protein interactions	JM Jordan	2023-01-01	Protein lysate RIPA buffer with protease inhibitor tablets Antibody (2 µg per reaction) Beads (50% slurry) PBS with 0.02% Tween Non-denaturing loading buffer (1X) LoBind tubes Liquid nitrogen	Prepare protein lysate (3600 µg protein) Preclear with beads Add antibody and incubate overnight at 4°C Add washed beads and incubate Wash beads with PBS-Tween Elute proteins with non-denaturing buffer Store samples at -80°C	This is a gentle protocol designed to preserve protein-protein interactions Uses minimal washing steps without extended rotations Includes preclearance step to reduce non-specific binding

#Protocol by JM Jordan 2023

1. Prepared a lysate solution with 3600 ug of protein for HFA and HFB

2. Adjusted to 1mL each with RIPA+Tablets

3. Precleared solution by adding 50ul of 50% beads to each tube of lysate

4. Aliquoted into 3 tubes/lysate (6 tubes total) and adjusted each to 1mL with RIPA+Tablets

5. Added 2 ug of antibody to each tube

6. Rotated overnight at 4C

7. RT rotation for 1h

8. Washed 600ul beads with PBS + 0.02% Tween

9. Resuspended beads in 600ul RIPA+Tabs

10. Added 100ul of bead solution to each tube

11. Rotated at RT for 1h

12. Washed beads 3X with 1ml PBS + 0.02% Tween with gentle pipet mixing (no 5 min rotation)

13. After final wash, resuspended beads in 200 ul PBS + 0.02% Tween and transferred to fresh LoBind tubes

14. Added 30 ul 1X Non denaturing loading buffer and eluted as usual at 37C for 5 min

15. Separated solution from beads into new LoBind tubes and snap froze in LN2 and stored at -80C.

16. Note: Freeze Input and leftover