Pierce BCA Assay problem - Protein values bizarre (Jun/15/2011 )
Hi,
I am doing a BCA Assay to quantify protein at different steps of purification.
I am quantifying proteins in a Ion Exchange Chromatography. The proteins are eluted in 100mM Tris and differnt conc of NaCl, starting at 150mM and going to 2M NaCl. I use my sample in 2 different concentrations to do the BCA: a 1:2 and a 1:5. When I look at the data, my protein amounts don't add up. Both the dilutions suggest a different amount of the same protein. What might be wrong?
do you blank for the buffer? tris will have an effect on the assay.
predoc on Thu Jun 16 00:53:22 2011 said:
Hi,
I am doing a BCA Assay to quantify protein at different steps of purification.
I am quantifying proteins in a Ion Exchange Chromatography. The proteins are eluted in 100mM Tris and differnt conc of NaCl, starting at 150mM and going to 2M NaCl. I use my sample in 2 different concentrations to do the BCA: a 1:2 and a 1:5. When I look at the data, my protein amounts don't add up. Both the dilutions suggest a different amount of the same protein. What might be wrong?
Usually we do our samples with a set of standards. The linear relationship between OD and protein concentration is followed only in a range of protein concentration. The protein concentration in your samples has to be in that range.
Hi,
I have a very urgent problem with the Pierce kit. My diluted standards show extremely high ODs and it's impossible to generate a standard curve. I use the normal Laemmli buffer (without bromphenolblue) and no substance should interfere...
Thx!