# BCA Assay Trial 2

## If at first you don’t succeed, trial trial again.

Happy #MicroplateMonday! With a working microplate reader available to us, we proceeded (again) with the BCA Assay. We followed the protocol generated during our last attempt. The only difference is that I broke the bottom of microplate well E9. Therefore, sample O07 only had two replicates (E7 and E8).

**Some photos from our lab work**:

**Figure 1**. Here I am pipetting 10 µL of my sample for the microplate.

**Figure 2**. Laura using a multipipetter to add 200 µL of our BCA working reagent to each well.

**Figure 3**. Our completed microplate! Well A1 is in the top left corner. Microplate contents specified here.

**Once in the Genome Sciences Building, we did the following**:

- Covered plate with film to prevent solutions from evaloprating
- Using an Eppendorf ThermoMixer C, we incubated our plate for 30 minutes at 37ºC
- Vortexed plate to mix solutions thoroughly
- Centrifuged plate for approximately one minute at 1500 rpm using Beckman-Couter centrifuge
- Used Labsystems Multiskan MCC/340 to read plate at 540 nm
- Opened Ascent Software Version 2.6 on accompanying computer
- Read microplate

**Calculations**

In order to start the Mini-Trypsin digestion, we need to know the volume of our sample that contains 30 µg of protein. To calculate this volume, we used the following steps.

- Calculate average wavelength for each standard and unknown samples using wavelength for each replicate
- Substract average absorbance for the blank standards from the average absorbance of the remaining standards and unknown samles
- This is the
**blank-corrected absorbance** - Create a scatterplot for the standards
- y-axis: BSA concentration (µg/µL)
- x-axis: blank-corrected absorbances (nm)
- Add polynomial trendline
- Display equation and R-squared value
- Using the trendline equation in the scatterplot, calculate protein concentration from absorbance
- Multiply each concentration by three
- We do this because we diluted our sample 1:2 with 50 mM NH4HCO3 in 6M Urea
- For each sample, calculate volume (µL) needed for 30 µg protein
- Need 100 µL to start Mini-Trypsin digestion
- Calculate how much 50 mM NH4HCO3 in 6M Urea needed for each sample to reach 100 µL volume
- 100 µL total volume - volume for 30 µg protein

The scatterplot I generated and a table of calculations are below:

**Figure 4**. Scatterplot of BSA concentration (µg/µL) versus blank corrected absorbances (nm). The trendline equation, y = 0.6633x^{2} + 1.3975x + 0.0049, was used to calculate protein concentration from absorbances.

**Table 1**. Calculations for each sample. Volume of samples and 50 mM NH4HCO3 in 6M Urea needed for 100 µL total volume based on having 30 µg of protein. *Note: “O124” should read “0127” under the column “Sample”*

Check back Tuesday for our Mini-Trypsin digestion! #TrypsinTuesday #MakeEverythingAHashtag