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Interference in a Pierce uBCA Assay with HIS-tagged protein

Interference in a Pierce uBCA Assay with HIS-tagged protein - Protein Science

Interference in a Pierce uBCA Assay with HIS-tagged protein - Post Questions and Discuss Protein Structure and Function, Protein Localization etc.. Please do not post Proteomic questions here, there is a Proteomic Forum below.


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Old 06-23-2010, 10:05 PM
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Default Interference in a Pierce uBCA Assay with HIS-tagged protein



Working under a denaturation protocol for a recombinant 6xHIS-Tagged protein.
Everything is at pH 8
Protein is solubilized in : 8M Urea, 20mM Tris, 500mM NaCl, 1% Triton X-100, 1mM b-ME
Resin is GE IMAC FF6 charged with NiSO4
Equilibration Buffer - 6M Urea, 20mM Tris, 1M NaCl, 1mM b-ME, 5mM Imidizole
Elution Buffer - 6M Urea, 20mM Tris, 1M NaCl, 1mM b-ME, 250mM Imidizole.

Here's my question:
When I perform a uBCA the values obtained are quite good, nice and high. The next step is dialysis or TFF against 6M Urea, 20mM Tris to go on to a Q anion exchange column. I perform a micro BCA following dialysis and I lost at least half of my protein...According to the BCA. I have done quanitative gel analysis and the quantity of the protein is the same before and after the dialysis so clearly my assay is to blame.
So what could be giving me the high reads on the uBCA assay? I perform dilutions enough so it can't be the Urea, NaCl, or Imidizole (1/32,1/64,1/128 in PBS)
The only thing I can come up with is that nickel is leaching off of the column.
Does anyone know how this can affect the uBCA assay or the chemistry behind how the Ni over takes the Cu?
Another possible solution is aggregates of my protein which I know it loves to do.
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assay , histagged , interference , pierce , protein , ubca


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