BCA results

11/23/2016

Today we completed the protein assay!

We followed the protocol I have written up here

*For Sample #56 we made a mistake and only have two replicates instead of three.

After I got the resulting absorbances from the Spectrophotometer plate reader I calculated the protein concentrations and determined how much of each sample I will need for the digestion protocol.

graph

1) Obtained measured wavelengths at 562nm for each of the standards and replicates from the spectrophotometer. I averaged the three replicates for each.

2) Subtracted the average blank measured absorbance at wavelength 562nm from the averages of the standards and unknown samples. This is the Blank-corrected Absorbance.

3) I created a scatter plot in excel for the standards by plotting the BSA concentration (ug/ul) on the Y-axis and the blank-corrected absorbances on the X-axis. I added a polynomial trendline and displayed the equation and r-squared value. I did those so I could easily plug in the X (absorbance) and calculate the Y (protein concentration).

4) Because I diluted with 1:2 with 50 mM NH4HCO3 in 6M Urea, I multiplied the protein concentrations I calculated by 3.

5) Then I calculated how much volume is needed to have 100ug of protein in each of my samples. Because we want to start the Mini-Trypsin digestion with 100ul, I figured out how many microliters of 50mM NH4HCO3 in 6M urea I needed to add.

see nice table

Well Contents Rep Abs. at 562nm Avg. Abs. Blank-corr. Abs. BSA conc. (ug/ul) Corr. for dilution of NH4HCO3 ul of sample required for 100ug ul to add of NH4HCO3
A1 Vial B 1 0.986 0.981 0.881 1.500 N/A N/A N/A
A2 Vial B 2 0.991 N/A N/A N/A N/A N/A N/A
A3 Vial B 3 0.966 N/A N/A N/A N/A N/A N/A
A4 Vial C 1 0.717 0.705 0.605 1.000 N/A N/A N/A
A5 Vial C 2 0.72 N/A N/A N/A N/A N/A N/A
A6 Vial C 3 0.679 N/A N/A N/A N/A N/A N/A
A7 Vial D 1 0.554 0.563 0.463 0.750 N/A N/A N/A
A8 Vial D 2 0.577 N/A N/A N/A N/A N/A N/A
A9 Vial D 3 0.558 N/A N/A N/A N/A N/A N/A
A10 Vial E 1 0.421 0.434 0.333 0.500 N/A N/A N/A
A11 Vial E 2 0.431 N/A N/A N/A N/A N/A N/A
A12 Vial E 3 0.449 N/A N/A N/A N/A N/A N/A
B1 Vial F 1 0.276 0.268 0.168 0.250 N/A N/A N/A
B2 Vial F 2 0.267 N/A N/A N/A N/A N/A N/A
B3 Vial F 3 0.262 N/A N/A N/A N/A N/A N/A
B4 Vial G 1 0.177 0.177 0.077 0.125 N/A N/A N/A
B5 Vial G 2 0.176 N/A N/A N/A N/A N/A N/A
B6 Vial G 3 0.179 N/A N/A N/A N/A N/A N/A
B7 Vial H 1 0.157 0.146 0.046 0.025 N/A N/A N/A
B8 Vial H 2 0.132 N/A N/A N/A N/A N/A N/A
B9 Vial H 3 0.15 N/A N/A N/A N/A N/A N/A
B10 Vial I 1 0.1 0.100 0.000 0.000 N/A N/A N/A
B11 Vial I 2 0.1 N/A N/A N/A N/A N/A N/A
B12 Vial I 3 0.101 N/A N/A N/A N/A N/A N/A
C1 Sample 1 1 0.838 0.817 0.717 1.195 3.584 27.9 72.1
C2 Sample 1 2 0.822 N/A N/A N/A N/A N/A N/A
C3 Sample 1 3 0.791 N/A N/A N/A N/A N/A N/A
C4 Sample 3 1 0.561 0.572 0.471 0.755 2.266 44.1 55.9
C5 Sample 3 2 0.572 N/A N/A N/A N/A N/A N/A
C6 Sample 3 3 0.582 N/A N/A N/A N/A N/A N/A
C7 Sample 4 1 0.719 0.743 0.642 1.059 3.176 31.5 68.5
C8 Sample 4 2 0.753 N/A N/A N/A N/A N/A N/A
C9 Sample 4 3 0.756 N/A N/A N/A N/A N/A N/A
C10 Sample 8 1 0.964 0.998 0.898 1.536 4.608 21.7 78.3
C11 Sample 8 2 0.977 N/A N/A N/A N/A N/A N/A
C12 Sample 8 3 1.053 N/A N/A N/A N/A N/A N/A
D1 Sample 11 1 0.619 0.590 0.490 0.787 2.361 42.3 57.7
D2 Sample 11 2 0.581 N/A N/A N/A N/A N/A N/A
D3 Sample 11 3 0.57 N/A N/A N/A N/A N/A N/A
D4 Sample 12 1 0.911 0.957 0.856 1.457 4.371 22.9 77.1
D5 Sample 12 2 0.989 N/A N/A N/A N/A N/A N/A
D6 Sample 12 3 0.97 N/A N/A N/A N/A N/A N/A
D7 Sample 16 1 0.799 0.790 0.690 1.146 3.437 29.1 70.9
D8 Sample 16 2 0.784 N/A N/A N/A N/A N/A N/A
D9 Sample 16 3 0.788 N/A N/A N/A N/A N/A N/A
D10 Sample 19 1 0.848 0.907 0.806 1.362 4.086 24.5 75.5
D11 Sample 19 2 0.892 N/A N/A N/A N/A N/A N/A
D12 Sample 19 3 0.98 N/A N/A N/A N/A N/A N/A
E1 Sample 20 1 0.747 0.750 0.650 1.073 3.218 31.1 68.9
E2 Sample 20 2 0.771 N/A N/A N/A N/A N/A N/A
E3 Sample 20 3 0.733 N/A N/A N/A N/A N/A N/A
E4 Sample 24 1 0.66 0.662 0.562 0.914 2.742 36.5 63.5
E5 Sample 24 2 0.681 N/A N/A N/A N/A N/A N/A
E6 Sample 24 3 0.645 N/A N/A N/A N/A N/A N/A
E7 Sample 27 1 0.785 0.785 0.684 1.135 3.406 29.4 70.6
E8 Sample 27 2 0.766 N/A N/A N/A N/A N/A N/A
E9 Sample 27 3 0.803 N/A N/A N/A N/A N/A N/A
E10 Sample 28 1 0.807 0.844 0.744 1.245 3.736 26.8 73.2
E11 Sample 28 2 0.856 N/A N/A N/A N/A N/A N/A
E12 Sample 28 3 0.87 N/A N/A N/A N/A N/A N/A
F1 Sample 32 1 0.827 0.829 0.728 1.216 3.649 27.4 72.6
F2 Sample 32 2 0.797 N/A N/A N/A N/A N/A N/A
F3 Sample 32 3 0.862 N/A N/A N/A N/A N/A N/A
F4 Sample 35 1 0.617 0.642 0.541 0.878 2.634 38.0 62.0
F5 Sample 35 2 0.651 N/A N/A N/A N/A N/A N/A
F6 Sample 35 3 0.657 N/A N/A N/A N/A N/A N/A
F7 Sample 36 1 0.974 0.979 0.879 1.500 4.501 22.2 77.8
F8 Sample 36 2 0.954 N/A N/A N/A N/A N/A N/A
F9 Sample 36 3 1.01 N/A N/A N/A N/A N/A N/A
F10 Sample 40 1 0.93 0.999 0.898 1.537 4.612 21.7 78.3
F11 Sample 40 2 1.024 N/A N/A N/A N/A N/A N/A
F12 Sample 40 3 1.042 N/A N/A N/A N/A N/A N/A
G1 Sample 43 1 0.589 0.572 0.472 0.756 2.267 44.1 55.9
G2 Sample 43 2 0.574 N/A N/A N/A N/A N/A N/A
G3 Sample 43 3 0.553 N/A N/A N/A N/A N/A N/A
G4 Sample 44 1 0.952 0.955 0.854 1.453 4.359 22.9 77.1
G5 Sample 44 2 0.92 N/A N/A N/A N/A N/A N/A
G6 Sample 44 3 0.992 N/A N/A N/A N/A N/A N/A
G7 Sample 48 1 0.948 0.957 0.857 1.458 4.374 22.9 77.1
G8 Sample 48 2 0.956 N/A N/A N/A N/A N/A N/A
G9 Sample 48 3 0.968 N/A N/A N/A N/A N/A N/A
G10 Sample 51 1 0.789 0.802 0.701 1.166 3.499 28.6 71.4
G11 Sample 51 2 0.779 N/A N/A N/A N/A N/A N/A
G12 Sample 51 3 0.837 N/A N/A N/A N/A N/A N/A
H1 Sample 52 1 0.902 0.889 0.789 1.329 3.986 25.1 74.9
H2 Sample 52 2 0.893 N/A N/A N/A N/A N/A N/A
H3 Sample 52 3 0.872 N/A N/A N/A N/A N/A N/A
H4 Sample 56 1 1.095 1.103 1.002 1.740 5.219 19.2 80.8
H5 Sample 56 2 1.11 N/A N/A N/A N/A N/A N/A
H6 Sample 56 3 omit N/A N/A N/A N/A N/A N/A
Written on November 23, 2016