8 replies to this topic

[Rchqing]

Hi, everyone!

I purified a protein several days ago and measured the concentration of the sample yesterday using two different method. First, it was measured by BCA kit. the concentration is 7.6mg/ml. Then I measured UV280 of the sample and the concentration was calculated to be 14.3mg/ml(I know the Ext. coefficient). This is very strange! I don't know which method is more convincible. Which method do you usually use for protein concentration measurement?

I am looking forward to your replies. Thank you！

[mdfenko]

factors that could affect your protein determination:

standard protein used to calibrate the bca

buffer in which the protein is solubilized

absorbance reading (linear range of the assay, dynamic range of the spectrophotometer)

dilution of the sample (to stay within effective range)

purity of the protein

[DRT]

My money would be on the BCA reading.   It would be unusual for BCA to give a dramatically low reading unless you have a copper chelator present.

[Smog187]

If you know the amino acid sequence then I would trust the Native scan. You could determine the concentration by denaturing your protein with some NaOH and using the extinction coefficient of your protein with the Tyrosines and Tryptophans at 260nm (if you have these amino acids).

[mdfenko]

Smog187, on 13 September 2012 - 06:32 PM, said:

If you know the amino acid sequence then I would trust the Native scan. You could determine the concentration by denaturing your protein with some NaOH and using the extinction coefficient of your protein with the Tyrosines and Tryptophans at 260nm (if you have these amino acids).

that should be 280nm

[Smog187]

mdfenko, on 14 September 2012 - 11:26 AM, said:

Smog187, on 13 September 2012 - 06:32 PM, said:

If you know the amino acid sequence then I would trust the Native scan. You could determine the concentration by denaturing your protein with some NaOH and using the extinction coefficient of your protein with the Tyrosines and Tryptophans at 260nm (if you have these amino acids).

that should be 280nm

Yeah! Thanks for catching that!

[Rchqing]

Smog187, on 13 September 2012 - 06:32 PM, said:

If you know the amino acid sequence then I would trust the Native scan. You could determine the concentration by denaturing your protein with some NaOH and using the extinction coefficient of your protein with the Tyrosines and Tryptophans at 260nm (if you have these amino acids).

Thank you very much! Could you give some suggestions about how much NaOH is needed?

[Smog187]

Rchqing, on 14 September 2012 - 09:39 PM, said:

Smog187, on 13 September 2012 - 06:32 PM, said:

If you know the amino acid sequence then I would trust the Native scan. You could determine the concentration by denaturing your protein with some NaOH and using the extinction coefficient of your protein with the Tyrosines and Tryptophans at 260nm (if you have these amino acids).

Thank you very much! Could you give some suggestions about how much NaOH is needed?

We use a final NaOH concentration of 0.1N in the cuvette. Also, make sure the cuvette you're using is designed to be used in the UV range.

[Rchqing]

Smog187, on 16 September 2012 - 04:32 PM, said:

Rchqing, on 14 September 2012 - 09:39 PM, said:

Smog187, on 13 September 2012 - 06:32 PM, said:

If you know the amino acid sequence then I would trust the Native scan. You could determine the concentration by denaturing your protein with some NaOH and using the extinction coefficient of your protein with the Tyrosines and Tryptophans at 260nm (if you have these amino acids).

Thank you very much! Could you give some suggestions about how much NaOH is needed?

We use a final NaOH concentration of 0.1N in the cuvette. Also, make sure the cuvette you're using is designed to be used in the UV range.

OK！ Thank you very much!