The main purpose of these slides is to convey information to the Professors, Lecturers, and Students. These slides contain authentic information about this topic which is mentioned in that.

1.  Protein Quantification by
Bradford’s method:

a
M.Phool Badshah

2. Estimation of Protein by Bradford’s method: `
Aim:
To determine the amount of protein in a given
sample by Bradford’s method.
Principle:
The dye coomassie brilliant blue G-250 (wavelength (max)=
465nm) appears as a pale-orange red protonated form (i.e), in
acid solution. It binds strongly to positive charged groups of
protein and also to hydrophobic regions in protein. As a result,
Blue colour is formed with a wavelength (max) at 595nm ((( on
binding to protein the wavelength (max) is shifted from 465 to
595nm))).

3. Materials:
 Bovine serum albumin (BSA).
 Co-omassive Brilliant blue dye.
 0.15 M NaCl
 Spectrophotometer .
 Tubes.
 Micropipettes.
 Distilled water.

4. Procedure:
 Standard Assay:
 Prepare a series of protein standards using BSA diluted
with 0.15M NaCl to final concentrations of 0 (blank = NaCl
only) 250, 500, 750, and 1500 micro-gram BSA /ml. Also
prepare serial dilutions of the unknown sample to be
measured.
 Add 100 microliter of each of the above to a separate test
tube .
 Add 5ml of coomassive blue to each tube and mix by
vortex.

5. a
 Adjust the spectrophotometer to a wavelength of 595nm,
and blank using the tube from step 3, which contain 0 BSA
 Wait 5 minutes and read each of the standards and each of
the sample at a 595-nm wavelength.
 Plot the absorbance of the standards versus their
concentration. Compute the extinction co-efficient and
calculate the concentrations of the unknown samples.

6. Microassay method:
 Prepare standard concentrations of BSA of 1, 5, 7.5, and 10
microgram /ml. Prepare a blank of NaCL only. Prepare a
series of sample dilutions.
Add 100 micro-litter of each of the above to separate
tubes (use microcentrifuge tubes). Add 1 ml of
Coomassive blue to each tube.
Turn on and adjust a spectrophotometer to a
wavelength of 595 nm and, blank the
spectrophotometer using 1.5 ml cuvettes.
.

7. a
Wait 2 minutes and read absorbance of each standard
and sample at 595nm.
Plot the absorbance of the standards versus their
concentration. Compute the extinction co-efficient
and calculate the concentrations of the unknown
samples.