|Register||Search||Today's Posts||Mark Forums Read|
|Protein Science Post Questions and Discuss Protein Structure and Function, Protein Localization etc.. Please do not post Proteomic questions here, there is a Proteomic Forum below.|
| ||LinkBack||Thread Tools||Display Modes|
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.
|assay , histagged , interference , pierce , protein , ubca|
|Thread||Thread Starter||Forum||Replies||Last Post|
|Human Cytome Project - Update 24 Jan. 2005||Peter Van Osta||Cell Biology and Cell Culture||1||08-01-2010 02:18 PM|
|Human Cytome Project - Update 6 Jan. 2005||Peter Van Osta||Cell Biology and Cell Culture||0||01-06-2005 10:18 AM|
|New Saccharomyces Sequences 11/27/04||Mike Cherry||Yeast Forum||0||11-28-2004 11:39 PM|
|New Saccharomyces Sequences 05/19/04||SGD Sequences||Yeast Forum||0||05-23-2004 04:06 PM|
|New Saccharomyces Sequences 09/10/03||SGD Sequences||Yeast Forum||0||09-11-2003 01:33 AM|