I trying to make protein-nanoparticle complex.
To estimate the amount of protein in the final sample, I know I have to calculate the conc of protein in supernatant.
(Initial conc of protien - conc of protein in supernatant)
but i don't know exactly how i calculate the amount of protein using above formula
Hi there,
If you know the sequence of the protein you can calculate its specific absorption coefficient at 280nm. Then a simple reading at 280nm for the starting solution and the final supernatant is enough. With a Nanodrop you may perform the reading with microvolumes.
The easiest approach to obtain "a number" for the total concentration of protein in a solution is to measure the Absorbance of the protein solution at 280 nm using a spectrophotometer. You first measure the absorbance of your buffer, repeat this with you initial protein sample and then repeat it with your final protein sample(take at least 3 measurements of each sample to obtain a more reliable average ... take more if you can). Subtract the buffer reading from your two protein readings to remove background absorbance caused by the buffer. Finally subtract your final protein reading from your initial protein reading to see the difference.
The absorbance reading in itself will only show you an indication of the difference in your TOTAL protein concentration. However, this number is NOT a concentration (which is generally provided in mg/ml or molarity). If you have only one protein in your sample and there are no other components that differ between the different samples, you can calculate the exact concentration in mg/ml or molarity by using the Extinction Coefficient of the protein in combination with the Absorbance reading. You can easily calculate the extinction coefficient of your protein using PROTPARAM from the EBI website by giving it the exact amino acid sequence of your protein. There are many on-line calculators that can then calculate the concentration for you if you have both numbers (absorbance and extinction coefficient). This will obviously not be reliable if you add other components (nucleic acid, peptide, antibody, etc...) to your final sample that affect the Absorbance and which are not present in the initial sample or the buffer.
Add known amount of protein. After centrifigation collect the supernatant of specific volume. Determine the amount of protein in it using some established assay method like such as UV absorbance, Bradford, BCA, Lowry assay, fluorescence-based method, SDS-PAGE, etc. Then extrapolate the protein amount to whole supernatant by considering Dilution factor.
Deduct the amount of protein in supernatant from amount added; and you will get the amount loaded in nanoparticle.
http://pubs.acs.org/doi/abs/10.1021/acs.analchem.5b01451
http://www.che.iitb.ac.in/courses/uglab/cl431/bl301-proteinassay.pdf
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