How to estimate milk protein by Bradford method

1. Presented By:
Deepanjan Basak
Roll No.L-15/FT/22

2. A whitish liquid containing protein, fats,
carbohydrates & various vitamins & minerals
that is produced by mammary glands of all
mature mammals after they have given birth
& serves as nourishment for their young.
What is milk?

3. Milk protein
casein whey protein other compounds
(peptides, lactoferrin, lysozome)
α β+ĸ ¥
α –lactalbumine β-lactoglobulin serum albumin immunoglobulin
Ig A Ig G IgM
Milk Protein Classification

4.  The Bradford protein assay is a spectroscopic analytical procedure (by
using Comassie Brilliant Blue G 250) used to measure the concentration
of protein in a solution.
 Fastest assay performed on milk proteins
 Can measure protein quantity as little as 1 to 20 µg
 Comassie dye can remain at room temperature for up to 2 weeks before
it start to degrade
Need for Bradford method

7. Metarials :
 Lyophilized bovine plasma gamma globulin
 Coomassie brilliant blue dye
 0.15 M Nacl
 Spectrophotometer & cuvettes
 Micropipettes

8. 1. Prepare a series of protein standard 0.15 M Nacl to final
conc. Of 0(blank = Nacl), 250, 500, 750 & 1500 µg/ml.Also
prepare serial dilution (2 times) of the unknown sample to
be measured.
2. Add 100 µl of each of the above to seperate test tubes.
3. Add 5.0 ml of coosammie blue to each test tubes & mix
vortex or inversion.
4. Adjust the spectrophotometer to a wavelength of 595 nm; &
blank using the tube which contain no protein.
5. Wait 5 min & read each of the standard & each of the
samples at 595 nm.
6. Plot the absorbance of the standard vs.Their conc.

10.  Concentration increase with the increase of
absorbance
 Protein quantity is directly proportional to the
change of colour(Reddish to Bluish).
 Activity of dye is reduced by the presence of
detergents, although this problem can be alleviated
by the addition of cyclodextrins to the assay mixture