BCA Assay Trial 1
Practice makes perfect?
Today we attempted a BCA Assay to quantify the amount of protein in our sample. However, the microplate reader in the Genome Sciences building wasn’t working! We’ll need to repeat this assay on Monday, Dec. 12, but here’s what we did:
The first step in our protocol involved making the necessary reagents.
BCA Working Reagent
- Got BCA kit
- Pipetted 20 mL of BCA Reagent A into a falcon tube
- Added 400 µL BCA Reagent B
- Vortexed to mix thoroughly
50mM NH4HCO3 solution
- Pipetted 5 mL nanopure water to a falcon tube
- Weighed 0.03953g of NH4HCO3 and added to nanopure
- Vortexed to mix thoroughly
- Poured contents of falcon tube into a graduated cylinder
- Pipetted nanopure water into the graduated cylinder until total volume was 10 mL
- Poured contents of graduated cylinder back into the falcon tube
- Vortexed to mix thoroughly
Lysis Buffer
- Pipetted 4 mL of 50mM NH4HCO3 solution into a new falcon tube
- Weighed 1.44g Urea and added to falcon tube
- Vortexed to mix thoroughly
- Poured contents of falcon tube into a graduated cylinder
- Pipetted nanopure water into the graduated cylinder until total volume was 6 mL
- Poured contents of graduated cylinder back into the falcon tube
- Vortexed to mix thoroughly
BCA Standards
Using the table below, we added a designated amount of the Lysis Buffer to a specified solution in snaptop centrifuge tubes. Pulse three times on vortex to mix thoroughly
Table 1. Volume of specified solution and Lysis Buffer added to each vial, along with resulting BSA concentration.
| Standard Vial | Solution | Lysis Buffer | BSA concentration |
|---|---|---|---|
| Vial B | 375 µL BCA Working Reagent | 125 µL | 1.5 µg/µL |
| Vial C | 325 µL BCA Working Reagent | 325 µL | 1.0 µg/µL |
| Vial D | 175 µL Vial B | 175 µL | 0.75 µg/µL |
| Vial E | 325 µL Vial C | 325 µL | 0.50 µg/µL |
| Vial F | 325 µL Vial E | 325 µL | 0.25 µg/µL |
| Vial G | 325 µL Vial F | 325 µL | 0.125 µg/µL |
| Vial H | 100 µL Vial G | 400 µL | 0.025 µg/µL |
| Vial I | N/A | 500 µL | 0 µg/µL |
Prepare samples for microplate
- I removed my snaptop centrifuge tubes labeled “11 µL” from the -80ºC freezer
- Placed samples in wet ice
- Added 22 µL of 50mM NH4HCO3 solution to each sample
- Vortexed and centrifuged each sample
- Returned samples to wet ice
After mixing all solutions, we obtained a 90 well microplate.
Creating the BCA Assay Microplate
We mapped out where each sample would go on the microplate in the table below, and pipetted accordingly. Each well contained 10 µL of the solutions designated in the table, as well as 200 µL of the BCA working reagent. The total volume in each well was 300 µL.
Table 2. Microplate arrangement.
| Well Number | Contents | Replicate |
|---|---|---|
| A1 | Vial B | 1 |
| A2 | Vial B | 2 |
| A3 | Vial B | 3 |
| A4 | Vial C | 1 |
| A5 | Vial C | 2 |
| A6 | Vial C | 3 |
| A7 | Vial D | 1 |
| A8 | Vial D | 2 |
| A9 | Vial D | 3 |
| A10 | Vial E | 1 |
| A11 | Vial E | 2 |
| A12 | Vial E | 3 |
| B1 | Vial F | 1 |
| B2 | Vial F | 2 |
| B3 | Vial F | 3 |
| B4 | Vial G | 1 |
| B5 | Vial G | 2 |
| B6 | Vial G | 3 |
| B7 | Vial H | 1 |
| B8 | Vial H | 2 |
| B9 | Vial H | 3 |
| B10 | Vial I | 1 |
| B11 | Vial I | 2 |
| B12 | Vial I | 3 |
| C1 | G10 | 1 |
| C2 | G10 | 2 |
| C3 | G10 | 3 |
| C4 | G18 | 1 |
| C5 | G18 | 2 |
| C6 | G18 | 3 |
| C7 | G48 | 1 |
| C8 | G48 | 2 |
| C9 | G48 | 3 |
| C10 | G58 | 1 |
| C11 | G58 | 2 |
| C12 | G58 | 3 |
| D1 | G68 | 1 |
| D2 | G68 | 2 |
| D3 | G68 | 3 |
| D4 | G77 | 1 |
| D5 | G77 | 2 |
| D6 | G77 | 3 |
| D7 | G92 | 1 |
| D8 | G92 | 2 |
| D9 | G92 | 3 |
| D10 | G97 | 1 |
| D11 | G97 | 2 |
| D12 | G97 | 3 |
| E1 | G119 | 1 |
| E2 | G119 | 2 |
| E3 | G119 | 3 |
| E4 | G131 | 1 |
| E5 | G131 | 2 |
| E6 | G131 | 3 |
| E7 | O07 | 1 |
| E8 | O07 | 2 |
| E9 | O07 | 3 |
| E10 | O15 | 1 |
| E11 | O15 | 2 |
| E12 | O15 | 3 |
| F1 | O37 | 1 |
| F2 | O37 | 2 |
| F3 | O37 | 3 |
| F4 | O47 | 1 |
| F5 | O47 | 2 |
| F6 | O47 | 3 |
| F7 | O55 | 1 |
| F8 | O55 | 2 |
| F9 | O55 | 3 |
| F10 | O77 | 1 |
| F11 | O77 | 2 |
| F12 | O77 | 3 |
| G1 | O107 | 1 |
| G2 | O107 | 2 |
| G3 | O107 | 3 |
| G4 | O119 | 1 |
| G5 | O119 | 2 |
| G6 | O119 | 3 |
| G7 | O127 | 1 |
| G8 | O127 | 2 |
| G9 | O127 | 3 |
| G10 | O142 | 1 |
| G11 | O142 | 2 |
| G12 | O142 | 3 |
| H1 | GBLANK | 1 |
| H2 | GBLANK | 2 |
| H3 | GBLANK | 3 |
| H4 | OBLANK | 1 |
| H5 | OBLANK | 2 |
| H6 | OBLANK | 3 |
We then covered the microplate and brought it over to the Genome Sciences building (and discovered the plate reader wasn’t working). We’ll have to repeat this entire protocol on Monday!