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IP data confusion

IP Western Blot bands

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#1 Biochem_newbie

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Posted 01 March 2013 - 01:41 PM

Hi there,

I am trying to see the interaction of Protein A (Myc tag) & Protein B (Flag tag)
I transfected in 293 cells, blotted to see whole cell protein expression and came out with the expected bands.

Next step was to IP.
I pre-cleared with Protein A/G beads for 1 hour, and then transferred the supernatant into new tubes and put Myc conjugated beads overnight.
Next day I washed with NP-40 three times and then prepped the beads with sample buffer and then boiled.

Ran a western and blotted with Flag. (IP with myc and blot with flag)
My samples were

empty vector (negative control), protein A, Protein B, and Protein A&B.
Whole cell expression was the same as before but in my IP sample lanes I see a band in lanes with Protein B (flag tag), and Protein A&B.
I know I should only see a band in the Protein A&B lane for my IP samples.

If someone could comment on this I would really appreciate it!

Thank you!

#2 bob1

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Posted 01 March 2013 - 01:48 PM

The problem seems to be that your flag tagged protein sticks to the myc beads - are your protein A/G beads on the same base as the myc one (e.g. both are sepharose)?

#3 Biochem_newbie

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Posted 01 March 2013 - 02:10 PM

Yes, both the protein A/G beads and the Myc conjugated beads are sepherose based

#4 bob1

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Posted 01 March 2013 - 06:29 PM

Have you tried washing more times ( and/or longer) or using more stringent washes (higher salt or stronger detergent)?




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