Thursday, April 19, 2007

To perform the estimation of protein by Biuret method

AIM: To perform the estimation of protein by Biuret method.

PRINCIPLE:
Biuret method is the simplest method for protein estimation. This method is sensitive to the amino acid composition of the protein. Its sensitivity is moderately constant from protein to protein and because of its simple procedure and quick result, it is used to estimate protein in crude extract over a large range of concentration. This method can also be used to monitor the concentration of protein during purification.
This assay is based on copper ions binding to peptide bonds of protein under alkaline conditions to give a violet or purple color. The intensity of the charge transfer absorption bond resulting from the Cu-protein complex is linearly proportional to the mass of protein present in the solution. The chromophore or light-absorbing center seems to be a complex between the peptide backbone and cupric ions.

PROCEDURE:
1) Pipette standard BSA (50 mg/ml) and test sample as indicated in table in duplicates.
2) Adjust the volume to 0.2 mi with distilled water.
3) Add 3 ml of Biuret reagent. Mix and incubate at Room Temperature for 10 minutes.Read the optical density using spectrophotometer at 600 nm or colorimeter using suitable filter and record the readings.

1) Construct a calibration curve by plotting average optical density reading on ‘Y’ axis against std. Protein concentration (in mg) on ‘X’ axis.
2) Record the value ‘X’ from the graph corresponding to the optical density reading for the test sample.
3) Calculate the sample concentration using the following formula.
Protein concentration in test Sample=(X/V)x1000 mg/ml

X= Value from graph in mg
V= Volume of Sample in ml
RESULT:

10 comments:

Ritambhara said...

thank you so much for putting up such a useful blog!!
its really great to see such posts at a blog which wud be helpful to entire biotech community!!

jawgie said...

hey thanks a lot man! i needed this! :D

sreya said...

thanx yar it really helped me.
am in 12th std and i needed this in biotech project

karan said...

THANKS BUT I DINT UNDERSTOOD THE ROLE
OF BSA

shreya said...

Thanks.Ur blog is nice.It really helps biotech students.

INDIA said...

tnx.that is so useful .
it would be better if u drew the graph too!

manishpratap said...

what is the volume of BSA and the sample amount ,you haven't quoted it.it would be great if you would let me know

sarika said...

thank u so much.its a very nice blog.thanks a lot

cynthya said...

thank yu sir!

ruchi said...

hey...thanks .....its of such a great help