I have been trying to Co-IP AR with a Peroxiredoxin unsuccessfully. I am working with a knockout cell line for negative control. Co-IP conditions are as follows:
Lysis buffer: 50mM TrisHCl pH 7.4
10mM NaCl
0.5% NP-40 (Nonidet P-40)
Halt Protease inhibitor cocktail
Wash Buffer: 25mM TrisHCl pH 7.4
150mM NaCl
0.1% NP-40 (Nonidet P-40)
1.Lyse 30mins on ice and centrifuge to pellet debri
2.Incubate 400ng of lysate with 1ug PRDX1antibody (30mins RT prior to beads or 1hr at 4degrees prior to beads)
3.Add 30ul previously blocked protein Gbeads and incubate1hr RT or 11 hr at 4degrees)
4.Spin down at 4degrees and wash twice
5.Elute with 2xLDS buffer to run on Bis-Tris gel system
I have heard that AR can easily precipitate out of solution and therefor it is better to not incubate overnight, hence why I started doing the RT incubations with rotation. The top and bottom blots show Co-IP with PRDX1 antibody. The Co-IP for top blots was using wash buffer for washes and bottom Co-IP use Lysis buffer for all steps.
My antibody does not seem to bind to PRDX1 during Co-IP but it works well for WB.
Any tips are greatly appreciated!
This link is useful
https://www.thermofisher.com/ar/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-protein-protein-interaction-analysis.html
Regards
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