I have a problem. I have purified a protein with method A and then B. After method A I have a protein concentration of 20 mg/ml and after method B a concentration of 18 mg/ml. Method B is dialysis and my sample was diluted, so the volume went from 5 mL (A) to 6 mL (B).

When I present my total amount of protein at these stages I have a higher amount of protein after step B (108 mg) than after step A (100 mg), which is ridiculous, just because of the dilution.

How should I think around this and present this data in my purification table? I know that there can't be more protein after the second step. Should I just calculate the amount of protein after step B, with the volume of step A? Since I know I haven't lost or gained any proteins during the dialysis, I have only gained more buffer which gives me a lower protein concentration.

Please, any advice is valuable to me! Thanks

  • 3
    $\begingroup$ What is method A? And obviously you've got a wrong concentration, because you did an error in your measurements or in your calculations. How shoud we know, we've seen neither. $\endgroup$
    – Karl
    Aug 13 '19 at 20:11
  • 1
    $\begingroup$ How are you determining protein concentrations? Repeat that measurement if possible on your final protein sample, and get an appropriate uncertainty estimate for this measurement. $\endgroup$
    – Buck Thorn
    Aug 13 '19 at 20:36

This is a great introduction to the concept of lack of precision (ie error) in measurements. It is unlikely that your volumes are exactly 5 mL and 6 mL or that your protein concentrations are exactly 20 mg/mL and 18 mg/mL. The errors in these measurements combine to give different calculated protein amounts even though the protein amounts are presumably the same (unless you've lost some protein in handling or your protein mass is low enough that some could pass through the dialysis membrane you are using). For example, if the actual values were A: 5.1 mL and 20.4 mg/mL and B: 5.9 mL and 17.8 mg/mL, you'd get the same value of 104 mg total protein for both.

So to answer your question about reporting, it's important to have a sense of how precise your measurements are. A common way to do this is to repeat the measurement multiple times and calculate a standard error. That way, you can report the value as something like 100 +/-10 mg, and you would expect that the range for the two samples would usually overlap. And next time, you might want to measure the volume and concentrations more precisely. Volume certainly should be easy to determine to at least the nearest 0.1 mL.


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