I have animal feed sample. I used 1 g sample and 5 ml lysis buffer to extract protein. I took 100 ul (10ul sample supernatant + 90 ul distilled water). After all the procedure i got concentration of protein that is 2428 ug/ml. After multiplying by dilution factor that is 10. Concentration will be 24280 ug/ml. How can i calculate percentage from it.
4/11/24
Dear Haseeb,
If I correctly understand your question, your extract contains 24,280 ug protein/mL. This is 24.28 mg/mL. Your total extract (5 mL) contains:
5 mL x 24.28 mg/mL = 121.4 mg or 0.1214 grams protein. This came from 1 gram of feed sample, so the % is 0.1214 g protein/1.0 g sample = 12.14% by weight.
I hope this information helps you. Good luck w/ your research.
Bill Colonna Dept. Food Science & Human Nutrition, Iowa State University, Ames, Iowa, USA [email protected]
W.J. Colonna Thank you. You solved my problem. I have one more question. When i make dilutions of sample 20 times and 40 times. Protein percentage varies. 20 times dilution have 11 % while 40 times dilution have 14 %. Because the sample is same so the protein percentage must be same. Please comment.
4/24/24
Dear Haseeb,
One of the difficulties in trying to answer your question is that I don't know what assay procedure you are using to measure protein. Is it a colorimetric assay, e.g., Bradford, BCA, DC/Lowry, etc.? Or are you doing it spectrophotometrically be measuring absorbance @ 280 nm?
If you are doing a colorimetric assay, what does your calibration curve look like? Is it linear? I assume you are preparing the dilutions correctly, so you are not experience a dilution error. I've done several types of colorimetric protein assays, such as the ones mentioned above. When you make a calibration curve, the comes a point when, with excess protein standard, the curve loses linearity (becomes curvi-linear). If the absorbance of your sample falls on the curved region of your calibration curve, the accuracy will suffer such that the reported value will be lower than the actual. If you test a smaller aliquot of the sample, or a more dilute sample, and the absorbance falls on the linear portion of the curve where quantitation is optimal, you will get an accurate result. The 1:20 dilution has twice the protein concentration of the 1:40 dilution. If you are using a colorimetric assay, is it possible that the amount of protein in the 1:20 dilution is still too high so that the absorbance falls on the non-linear portion of your clibration curve? This could explain the lower % you are seeing. Since the 1:40 dilution has half as much protein, then if its absorbance falls on the linear portion of the curve, you'll get an accurate answer. This may be part of the problem you have.
I hope this information helps you. Good luck w/ your research.
Bill Colonna Dept. Food Science & Human Nutrition, Iowa State University, Ames, Iowa, USA [email protected]
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