Myc tagged protein sticking to beads.

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Scarnati14
Scarnati14's picture
Myc tagged protein sticking to beads.

 I am attempting to co-immunoprecipitate a FLAG tagged protein with a myc tagged protein. I am puuling down the FLAG protein and probing for the myc one. My control is simply adding no Ab to one equivalent sample. My problem is the myc tagged protein is being pulled down even in my no Ab control. I use protein A beads and that is what the myc seems to be sticking to. I have tried many different buffers to incubate my IPs in as well as many different wash buffers for my beads. It still seems to stick! Does anyone have any advice and/or ideas?

Thanks
-Scar

Chin Fen Teo
Chin Fen Teo's picture
 Hi Scarnati14,

 Hi Scarnati14,

Are you using agarose or sepharose based beads? Have you tried to preclear your samples prior to your IP? It helps to minimize the non-specific binding. Even using magnetic beads from certain vendors, I will still suggest the preclearing step.

Also, what content of detergent you have in this experiment? Have small amount of detergent (says, 0.5-1% NP40) may also help to reduce the non-specific binding without disrupting the protein-protein interaction.

Last straw, you can always elute with Flag peptide instead of boiling. In this case, you don't really have to worry about eluting non-specific binding at all.

Good luck.