Hi all,
I'm trying to CoIP endogenous Miro1 and 2 proteins in HeLa cells. They are mitochondrial protein and isoform of each other. We suppose that Miro proteins interact with each other and I want to see If I can pull them down together or not. I made double tagged Miro1-GFP and Miro2-MYC stable HeLa cell line and I use MYC/GFP Agarose or Agarose magnetic beads to pull down those proteins together. When I use each of the MYC or GFP beads I can pull down the tagged protein easily with high efficiency but I can not detect other protein-interactor by western blotting. I have used different protocols. The first one is from Chromoteck and I used magnetic beads to pull down the protein. It's lysis buffer has 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5 %, Nonidet™ P40 Substitute, 0.09 % sodium azide and the washing buffer has 10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.05 % Nonidet™ P40, Substitute, 0.5 mM EDTA, 0.018 % sodium azide. I wash the beads three times with low salt(150mM) and last wash I increase the salt concentration to 500mMthe The second protocol comes from a colleague and he could pull down overexpressed Miro1 and Miro2 together in HEK cells and I use Chromoteck Agarose beads to pull down Miro proteins. The lysis buffer has 10mM Tris-HCl pH7.4, 150mM NaCl, 1.5mM MgCl2, 5m EDTA, 1% Triton, 10% glycerol, 1 mM NaF, 50mM Na2H2P2O7 and the washing buffer has 10mM TrisHCl pH 7.5, 500mM NaCl, 1% Triton, 0.5% NP-40, 1mM MgCl2, 1mM EDTA, 0.5mM EGTA. For both protocol I add protease inhibitor when I want to do CoIP. All the attempt I made haven't been successful in terms of pulling down the interactor proteins beside the main protein so far . I would appreciate if anyone shares it's experience in pulling down of endogenous proteins especially Miro proteins a have any recommendations for me to solve this problem and to improve my work.
Thank you so much in advance
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