Determining the protein content of each of your Mytilus gill tissue preparation fractions.
The Bradford Protein Assay is a dye-binding assay that is complete after 10 minutes. The dye binds the protein and shifts its absorbance upon binding.
In order to get the most accurate results in this timed assay you will need to partition three tasks between your partners.
Student #1: At 1 minute intervals add Bradford reagent to your samples and mix.
Student #2: Beginning at T=10:00 minutes, move the reagent-treated sample to the cuvette and put the cuvette in the spectrophotometer. Repeat for the next sample at T=11:00 minutes.
Student #3: Record the absorbance at 595 nanometers. Dump and rinse the cuvette and set it carefully on the bench for student #2.
But first there is a lot of work you must do to set up this assay, including complex and careful pipetting.
§ Make sure you have a separate labeled tube containing 2 mL of Homogenization Buffer (HB).
§ Make sure you have a couple of separately labeled tubes containing 3 mL Resuspension Buffer (RB).
§ Make sure you have a thawed and well-mixed eppendorf tube of 1 mg/mL Protein Standard (BSA).
§ Make sure you have your five thawed and mixed sample tubes from the Mytilus mitochondrial preparation.
§ Identify and know how to use the instruments at your station.
§ Make sure you have a p20, p200, and p1000 at your station along with sufficient yellow and blue tips.
§ Make sure you have a bottle of Bradford Reagent at your station. 50 mL of reagent should be enough with careful pipetting.
§ If using glass pipette, check that the 1 mL graduated glass pipette measures all the way to the tip. Then test the blue or green pumps, or rubber bulbs on your glass pipette using distilled water to make sure it won’t leak or drip. If using an autopipette with a blue tip, be sure to pipette slowly and carefully.
§ Number the 13 mm glass tubes from 1 to 41 and arrange in test tube rack with a space between groups of three tubes.
Tube # |
Volume of |
Buffer |
Volume of |
Sample |
@ time = |
Reagent |
Absorbance 595 |
||
0 |
100 uL |
HB |
+ |
0 |
0:00:00 |
1 mL |
|||
1 |
100 uL |
HB |
+ |
0 |
1:00:00 |
1 mL |
|||
2 |
100 uL |
HB |
+ |
0 |
2:00:00 |
1 mL |
|||
3 |
100 uL |
RB |
+ |
0 |
3:00:00 |
1 mL |
|||
4 |
100 uL |
RB |
+ |
0 |
4:00:00 |
1 mL |
|||
5 |
100 uL |
RB |
+ |
0 |
5:00:00 |
1 mL |
|||
6 |
95 uL |
RB |
+ |
5 uL |
Known Protein Standard (1mg/mL BSA) |
6:00:00 |
1 mL |
||
7 |
95 uL |
RB |
+ |
5 uL |
Known Protein Standard (1mg/mL BSA) |
7:00:00 |
1 mL |
||
8 |
95 uL |
RB |
+ |
5 uL |
Known Protein Standard (1mg/mL BSA) |
8:00:00 |
1 mL |
||
9 |
92.5 uL |
RB |
+ |
7.5 uL |
Known Protein Standard (1mg/mL BSA) |
9:00:00 |
1 mL |
||
10 |
92.5 uL |
RB |
+ |
7.5 uL |
Known Protein Standard (1mg/mL BSA) |
10:00:00 |
1 mL |
||
11 |
92.5 uL |
RB |
+ |
7.5 uL |
Known Protein Standard (1mg/mL BSA) |
11:00:00 |
1 mL |
||
12 |
90 uL |
RB |
+ |
10 uL |
Known Protein Standard (1mg/mL BSA) |
12:00:00 |
1 mL |
||
13 |
90 uL |
RB |
+ |
10 uL |
Known Protein Standard (1mg/mL BSA) |
13:00:00 |
1 mL |
||
14 |
90 uL |
RB |
+ |
10 uL |
Known Protein Standard (1mg/mL BSA) |
14:00:00 |
1 mL |
||
15 |
87.5 uL |
RB |
+ |
12.5 uL |
Known Protein Standard (1mg/mL BSA) |
15:00:00 |
1 mL |
||
16 |
87.5 uL |
RB |
+ |
12.5 uL |
Known Protein Standard (1mg/mL BSA) |
16:00:00 |
1 mL |
||
17 |
87.5 uL |
RB |
+ |
12.5 uL |
Known Protein Standard (1mg/mL BSA) |
17:00:00 |
1 mL |
||
18 |
85 uL |
RB |
+ |
15 uL |
Known Protein Standard (1mg/mL BSA) |
18:00:00 |
1 mL |
||
19 |
85 uL |
RB |
+ |
15 uL |
Known Protein Standard (1mg/mL BSA) |
19:00:00 |
1 mL |
||
20 |
85 uL |
RB |
+ |
15 uL |
Known Protein Standard (1mg/mL BSA) |
20:00:00 |
1 mL |
||
Tube # |
Volume of |
Buffer |
Volume of |
Sample |
|||||
21 |
90 uL |
HB |
+ |
10 uL |
Mussel Tissue Homogenate |
21:00:00 |
1 mL |
||
22 |
90 uL |
HB |
+ |
10 uL |
Mussel Tissue Homogenate |
22:00:00 |
1 mL |
||
23 |
90 uL |
HB |
+ |
10 uL |
Mussel Tissue Homogenate |
23:00:00 |
1 mL |
||
24 |
90 uL |
HB |
+ |
10 uL |
First Supernatant |
24:00:00 |
1 mL |
||
25 |
90 uL |
HB |
+ |
10 uL |
First Supernatant |
25:00:00 |
1 mL |
||
26 |
90 uL |
HB |
+ |
10 uL |
First Supernatant |
26:00:00 |
1 mL |
||
27 |
90 uL |
RB |
+ |
10 uL |
First Pellet |
27:00:00 |
1 mL |
||
28 |
90 uL |
RB |
+ |
10 uL |
First Pellet |
28:00:00 |
1 mL |
||
29 |
90 uL |
RB |
+ |
10 uL |
First Pellet |
29:00:00 |
1 mL |
||
30 |
90 uL |
RB |
+ |
10 uL |
Second Supernatant |
30:00:00 |
1 mL |
||
31 |
90 uL |
RB |
+ |
10 uL |
Second Supernatant |
31:00:00 |
1 mL |
||
32 |
90 uL |
RB |
+ |
10 uL |
Second Supernatant |
32:00:00 |
1 mL |
||
33 |
90 uL |
RB |
+ |
10 uL |
Second Pellet |
33:00:00 |
1 mL |
||
34 |
90 uL |
RB |
+ |
10 uL |
Second Pellet |
34:00:00 |
1 mL |
||
35 |
90 uL |
RB |
+ |
10 uL |
Second Pellet |
35:00:00 |
1 mL |
||
36 |
100 uL |
HB |
+ |
0 |
36:00:00 |
1 mL |
|||
37 |
100 uL |
HB |
+ |
0 |
37:00:00 |
1 mL |
|||
38 |
100 uL |
HB |
+ |
0 |
38:00:00 |
1 mL |
|||
39 |
100 uL |
RB |
+ |
0 |
39:00:00 |
1 mL |
|||
40 |
100 uL |
RB |
+ |
0 |
40:00:00 |
1 mL |
|||
41 |
100 uL |
RB |
+ |
0 |
41:00:00 |
1 mL |
|||