BCA Protein Determination Method
Main sample-Each group will prepare the protein extraction for one of the four
sample meats. And will measure the protein content using the table below. Each
group will share their protein extract with the other groups.
Secondary samples- Samples received from other groups will measure the
absorbance at ONE dilution.
Sample Preparation:
1. Weigh out about 90 g of meat and coarsely chop sample
2. Blend 1 part meat with 3 parts extraction buffer (i.e. 90g meat: 270ml
extraction buffer) for 1 minute in a blender
3. Pour 30 ml of homogenate into a 50ml labeled centrifuge tube
4. Centrifuge samples at 2000 x g for 15 minutes at room temperature
5. Collect supernatant
6. Filter the supernatant using Whatman filter paper in a funnel (moisten filter
paper with extraction buffer)
7. Cap filtered supernatant this is your prepared sample and will be used to
measure protein content
BCA Protein Assay
1. Prepare working reagent by combining Pierce reagent A with Pierce reagent
B, 50:1 (v/v) ***WE HAVE PREPARED THIS AHEAD OF TIME***
2. Prepare the following dilutions of the supernatant in extraction buffer
3. In test tubes, prepare duplicated of each reaction mixture of diluted extracts
and BSA solution standards (using 1 mg BSA /ml solution)
Tube
Identity
dd Water
(μl)
BSA Std.
(μl)
Meat
Extract (μl)
Blank
Std. 1
Std. 2
Std. 3
Std. 4
Std. 5
Sample 1:5
Sample 1:10
Sample 1:20
50
40
30
20
10
0
25
25
25
0
10
20
30
40
50
0
0
0
0
0
0
0
0
0
25
25
25
Working
Reagent
(ml)
1
1
1
1
1
1
1
1
1
4. Mix eact reaction mixture with a vortex mixer, then incubate in water bath at
37°C for 30 min
5. Read absorbance of each tube at 560nm using a spectrophotometer
6. Use the data from the BSA samples to create a standard curve of absorbance
at 562nm versus μg protein/50 μl. Determine the equation of the line for the
standard curve. Calculate the protein concentration (μg/ml) of the extract
from each meat sample using the equation of the line from the BSA standard
curve and the absorbance value for a dilution of the meat extract that had an
absorbance neat the middle point of the standard curve. Remember to
correct for dilution used.