3 replies to this topic

[predoc]

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?

[mdfenko]

do you blank for the buffer? tris will have an effect on the assay.

Edited by mdfenko, 15 July 2011 - 06:11 AM.

[qzlabs]

predoc, on 15 June 2011 - 04:53 PM, 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.

[Gerlinde]

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!