You would normally calculate the amount of protein in the reaction based on the amount you put in:

Suppose your stock solution was 1 mg/ml protein and you diluted it 1:1,000 into the reaction. Then the concentration in the reaction was  0.001 mg/ml or 1 µg/ml. Multiply by the reaction volume (in ml) to get the amount in µg. Multiply by 1000 to get the amount in nanograms.

Trying to measure the protein concentration in a reaction by absorbance is difficult because the protein is often very dilute, and other substances may be present that also absorb at the same wavelength (e.g. 280 nm) as the protein. You could use a colorimetric protein assay such as the Lowry, Bradford, or BCA method if the protein is concentrated enough and the reaction doesn't contain any interfering substances.

If the protein concentration in the reaction is too dilute and/or the reaction does contain interfering substances, you could precipitate the protein with trichloroacetic acid + sodium deoxycholate to concentrate it and remove contaminants, then use the colorimetric protein assay to measure the amount of protein. This requires at least a few micrograms, and a standard curve.

you could do immunoelectrophoresis (western blot) tagging your protein of interest with an antibody

Most assays to quantitate a protein are not sensitive enough at the ng level.  Performing quantitative Western blots is NOT trivial.  You must ensure that you are in the linear section of your antigen with respect to the ECL detection kit that you are using.  You will also need to ensure that your signal is NOT saturated and finally you will also need a purified protein control.

What do you mean about ``in a reaction`` are you talking about an enzyme reaction? Is your protein pure or part of a mixture of proteins?  If you want to quantitate an enzyme from a mixture of a proteins you may want to consider an active site titration with a specific inhibitor if available.

You could also use mass spectrometry which will provide you with the necessary sensitivity provided that you have a pure sample devoid of detergent or glycerol.

Hopes this helps

Marc

Thanks! And the reaction we are doing is to test the enzymatic cleavage activity of the RAG protein. We recently transfected 293T cells with RAG plasmid coding for a form of the protein with a MBP tag on the amino end. After harvesting this I wanted to know if the cleavage activity is comparable to our previous batch of protein. So I ran the reaction with DNA, 2ul of protein and some buffers and ran the results of this on a denaturing PAGE with urea. The activity of the protein was a bit higher than previous runs so I wanted to see if there was a way to quantify this to see if we have a higher concentration of protein. 

Kyle,

What is a bit higher?  You likely have a small increase in your protein expression probably within experimental error.  If you want to be thorough, I would use your tag and purify your protein which would allow you to determine the protein concentration.  Because of your tag you may NOT need to do chromatography, batch mode purification will likely be sufficient.

Marc

Providing your samples contain enough protein it may be easiest to perform a BCA assay using the Pierce 660nm reagent. Simply make a standard BSA curve (50-2000ug/mL) and then make a simple 1:1 dilution of your sample (or several dilutions if you wish)(the presence of up to 1% detergent does not cause problems either). The reagent will turn blue in proportion to the concentration of protein (which most nanodrop machines have a programme for measuring). Using the BSA standards it will compare your sample of interest and tell you the relative concentration. Prep time is only 10min and it's pretty accurate. You can easily find the upper and lower limits of accuracy for this assay online, including in nanodrop manuals.

Hope this helps

DMF