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IP no target protein in elution


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#1 chouchou

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Posted 03 March 2011 - 09:45 AM

Hi, I'm new in immunoprecipitation and just did one trial with Protein G Sepharose (Sigma), however I didn't see any target protein in my elution. What's wrong?
I used 20mM sodium phosphate (pH7.0) as my washing buffer, and 0.1M glycine-HCl (pH2.7) as eluting buffer. I'm using Anti-c-Myc (Sigma) as my AB, but not sure of how much should I use, so I added 2ul undiluted AB into 20ul resin. Does all this seem right? Thank you for help...

#2 Ketil.Pedersen

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Posted 21 December 2011 - 03:44 AM

Hi,

For magnetic beads 1-10µg Abs per 50µl beads.

A few suggestions
  • Verify binding/specificity of your antibody to your antigen, e.g., by ELISA.
  • Check the binding of Abs to the beads. If the Abs are not captured and bound to the beads,
  • If you have used the indirect method, try the direct method. Conversely, if you have used the direct method, try the indirect method.
  • Check the amount of beads and sample volume. increase the amount of beads or the concentration of your antibody during coupling.
  • Increase the incubation time.
  • Try another antibody.
*********************************
Ketil Winther Pedersen, Ph.D
Head of Technical support for Dynabeads products
Tel: +47 22 06 11 10 (direct)
Fax: +47 22 51 95 05
eurotech@lifetech.com
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