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Ganglioside GT1b binding assay - no binding of + control proteins and non-specif

Ganglioside GT1b recombinant His-tag

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#1 Bea Kerr

Bea Kerr


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Posted 22 February 2012 - 08:00 PM

Hi, I am trying to perform as GT1b binding assay to assess the binding ability of a His-tagged recombinant protein. I have performed the assay numerous times using 2 different GT1b and 3 different positive control proteins and EVERY time my His-tagged test protein and His-tagged negative control protein bind to the plate and the positive controls only show baseline readings.
I have tried coating the plate with and without GT1b and I get the same level of binding of my His-tagged proteins so I assume they are binding to the plate.

Wells coated with GT1b in MetOH and allowed to evaporate at RT overnight (or MetOH without GT1b for control plate), have also tried binding ganglioside in PBS/BSA and PBS/Skim
Block - have tried BSA and skim
Proteins in PBS/BSA/Tw20 or PBS/Skim/Tw20 - 37C for 2 h
Primary Ab (as above) - 1 h @ 37C
Secondary Ab (as above) - 1 h @ 37C
Develop (HRP)

It raises these questions:
1) Why aren't ANY the proteins binding to GT1b?
2) Why are the His-tagged proteins binding to the plate?
3) Why aren't the + control proteins at least binding to the plate (they are not His-tagged)

This leads me to believe that the His-tag is involved somehow, but I still don't understand why the positive controls aren't binding to the GT1b. Is the GT1b not coating onto the plate at all and being washed off? This is what prompted me to buy a new GT1b from a different company, but I still get the exact same result. Even if I (somehow) rectify the GT1b issue, why am I getting such high readings from the His-tagged proteins and how do I prevent this non-specific binding.

I am following a published protocol and I'm going a bit crazy because I don't know what I'm doing wrong and this is one of the last experiments I need to do to finish my PhD.
Any suggestions?

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