We have a calculation dilemma for a GH ELISA kit since this kit is designed for volume (liquids) (media or plasma) and we are measuring GH in cell extracts normalized by protein (micrograms of protein). We want to know how many nanograms (ng) of GH do we have in one milligram (mg) of total protein.
The standard curve is given in nanograms of GH / millilitre (mL), standards were prepared as follow: 10, 5, 2.5, 1.25, 0.62, 0.31 ng (GH) / millilitre (mL). Each plate-well was filled with 100 microliters (uL) of each standard.
Our sample was added to each plate-well at a concentration of 50 micrograms (ug) in 100 microliters (uL) (= 0.1 mL).
We got that our sample: 50 micrograms of protein (ug) contained in 100 microliters (uL) correspond to value of 1.004 GH ng per milliliter (mL) by direct interpolation to the standard curve .
According to our calculations we have 2.008 ng of GH / mg of protein.
Rationale: 1.004 ng of GH / mL = 0.1004 ng / 100 uL and if 50 ug of protein are contained in 100 uL then we have 0.1004 ng GH in 50 ug of protein. Finally, 20 x 50 ug = 1 mg therefore 20 x 0.1004 ng of GH = 2.008 ng of GH / mg of protein.
Are we correct?
To make the calculation easier to understand, eliminate the volume and just work in terms of ng. You can do this because the sample volume and standard volume were the same, 100 µl. The standards used for the standard curve contained 1, 0.5, 0.25, 0.125, 0.062, 0.031 ng of GH.
Your sample contained 0.100 ng GH based on the standard curve.
The sample volume was 100 µl, so the GH concentration was 1.00 ng/ml.
The total amount of protein in the sample was 50 µg, so the GH content was 20 ng/mg.
The standard curve contained according to the manual 10, 5, 2.5, 1.25, 0.62, 0.31 ng GH per mL. And we placed a 1/10 of that standard per well which is 1, 0.5, 0.25, 0.125, 0.062, 0.031 ng of GH / 100 ul. Interpolation gave us 1.004 ng GH / mL which equals: 0.1004 ng GH / 0.1 mL
So if we have 50 ug of protein/ 100 ul that means we have 500 ug of protein in 1 mL, or not?
I have been told exactly what you told me, "do not consider the volume", but this doesn't makes sense at all to me since it is not the same to have 50 ug protein in 100 ul and 50 ug of protein in 1 ml. So that volume difference should imply a correction, or why not?
I have been breaking my head all day long with this "not volume consideration" suggestion.
The presentation of your situation is very complicated, but calculation should be very simple at the end. If you use the concentration values of the standards (ng/ml) and calculate the concentration value for the sample, you dont have to use the volumes. And you dont have to use total amounts. Just use concentrations for everything, its simpler.
Does your sample produce a signal between those of the standards prepared at 0.62 and 1.25 ng/ml? And when you calculate the exact concentration in your sample is 1.004 ng/ml? Is that right?
If so, as you have 1.004 ng/ml of GH and 0.5 mg/ml of total protein, which indeed means 2.008ng GH/mg total protein.
If there were other dilution steps that you didnt mention, the results could vary.
Gracias Gertrudis, yes, it was a very simple situation that turned very complicated.
No other dilution steps were included.
Sorry about the dumb mistake in my answer. It should have been 2 ng/mg, of course.
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