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I will measure absorbance for measurement of protein concentration.

The sample solutions are prepare in serial dilutions to the volume of 100 uL to obtain a certain concentration of the protein in the buffer, lets say 2 mg/mL, then 20 uL is added to the cuvette with 1 mL Bradford reagent.

Now my question is, is the protein concentration still 2 mg/mL, I mean, I know it is not in the cuvette with the reagent added, but do I have to take this into account when measuring the absorbance? Or should I recalculate the dilutions to get the concentration 2 mg/mL in total with the reagent added?

This might be a dumb question but the hour is late and my brain is scrambled eggs.

Thank you in advance

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  • $\begingroup$ The absorbance value you are getting is due to the protein content in the cuvette. So, your calculation is based on how you plot your calibration curve. Then, you have to back calculate according to your dilution. $\endgroup$ – Mathew Mahindaratne Jul 5 at 2:08
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should I recalculate the dilutions to get the concentration 2 mg/mL in total with the reagent added?

Either way works as long as you are consistent.

Plot dilute concentration

You can use the final concentration in the cuvette and plot this against the measured absorbance. When you use this standard curve, it will also give you concentration of your unknown in the cuvette. To get the undiluted concentration of the unknown, you have to do one extra step, multiplying the concentration in the cuvette by the dilution factor.

Plot undiluted concentration

You can use the undiluted standard concentrations and plot this against the measured absorbance. When you use this standard curve, it will give you the undiluted concentration of your unknown. This only works if you always dilute by the same factor.

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