Question

So this is probably a really silly question but I'm stuck...

Suppose you have the following egg-white sample A

Volume of sample A = 5 ml

Preparation of a Biuret reaction (sample B):

0.1 ml of Sample A

0.9 ml water

4.0 ml Biuret reagent

Abs of the reaction product (sample B) at 540 nm =0.200

Using YOUR OWN data, estimate how many total mg of protein was present in sample A. Note, I asked for the amount of my in the starting sample, not the number of mg in the cuvette.

How do I do this? I have all sorts of data, and I don't know what to use to even start this. The problem says to calculate the mg/mL in the cuvette from absorbance, but I don't have an epsilon value so I don't know how to go about doing this.

So I have a 10 mg/mL solution of BSA which is what we used for the 540 nm part of this experiment. I also calculated an experimental molar extinction coefficient of 70.268 for 280 nm. Is this enough info?

Answer 1

If you have plotted the standard curve for BSA where you have taken the different concentration of BSA. In the standard curve, each mg of protein corresponds to the OD. With the help of BSA standard curve, you can measure the concentration of protein.

In case of Beer-Lambert law

A= Cl

Here A = OD
= Molar extinction coefficient

l = path length which is normally 1 cm

A= 0.2

= 70.268

l = 1cm

SO C= 0.2 /70.268 *

0.00284 Molar

Again we need to find its MW to find out the concentration

value also not seem correct. value depends on the number of tyrosine, tryptophan and cysteine residue.

at 280 = # tyrptophan *5500 + # tyrosine *1490 # cysteine residue *125

Your molar extinction coefficient is less than the value of cysteine residue.

If you have BSA standard curve then you can easily calculate the concentration of egg-white protein.