The Bradford Assay

The purpose of the Bradford Assay is to measure an unknown mass of protein in solution. First you will prepare a series of different BSA standard solutions and your sample, then you will add the Bradford reagent (Coomassie Brilliant Blue dye), and finally measure the absorbance of these solutions. Next you will create an “Absorbance vs. Protein Mass” standard curve. This curve will then be used to analyze unknown protein concentrations based on their measured absorbances.

Outline

Walkthrough

1. Spectrophotometer Warm-Up:  Here’s the Genesys20 spectrophotometer from Thermo Spectronic

2. Prepare samples & mix em up:  Here’s most of the stuff. 

Pipette your choice of seven volumes (0≤volume≤60µL; try to cover the entire range) of 1μg/μL BSA solution into separate test tubes (use the tubes shown on your right- they’re large enough to accommodate the 3.00mL of Bradford reagent you will add later).  Also pipette the assigned volumes of unknown into respective test tubes.  Add 3.00mL of Bradford reagent to each tube- use the 1000µL pipette to dispense three 1mL portions (this volume should be as exact as possible, while the added volume of protein solution can vary because it is much smaller than 3.00mL & will not alter the solution volume or concentration very much).  After adding  the Bradford reagent, immediately cover the tube with parafilm & mix it by inversion (do not wait for all tubes to be prepared!).  Then let the tubes sit for ≥10min.- once their color develops it will be stable for over an hour. 

3. Measure those absorbances:  The cuvette has two different lengths of solution (you can see how the edges are tapered towards the bottom of the side facing you); if you don’t measure with the same orientation each time you will not be able to analyze the results without additional tedious calculations & guesswork.      

Close the door on the spectrophotometer when measuring the absorbances.  To add solutions to the cuvette-  place~3mL of the most dilute BSA solution in a cuvette & measure its absorbance.  When finished, remove this sample (via pasteur pipette) & measure the absorbance of the next most dilute BSA solution.  Repeat this until all BSA absorbances have been measured, & then repeat to measure the unknown absorbances (from most to least dilute- if applicable). 

4. Clean up after yourself:  Dispose of all solutions in hazardous waste container & clean out the cuvette.

5. Calculate your protein concentration:

Excel will automatically consider the column on your left to be the independent variable in scatter plots, which you’re gonna make next. (Note: this data is mad fake).

A plot of your data should appear.

This “Format Trendline” box will appear after you click “Add Trendline…”

The “Linest” function in Excel can approximate, better than the approximation made on your graph, a linear function to your data. 

The “function arguments” box will pop up.  Place your cursor in the appropriate box before highlighting the cells.

If you are using a Mac, Command+Shift is the equivalent to Control+Shift+Enter in Windows.

You will need to record some of these numbers in your lab report.  Make your graphs according to journal standards in your report. 

Next, you must estimate the amount of protein in your unknown samples from the absorbance data you measured for them.

Beers law describes the relationship between absorbance & concentration:

absorbance = εlc (where ε = extinction coefficient, l = length of solution in cuvette, & c = concentration).

This equation can be rearranged to give concentration in terms of absorbance.

→ →

In your experiment, volume, ε, and l are constants - so this equation can be simplified to

To find a linear equation that approximates this relationship, you use the linest function as you did before, except this time select the protein concentration data for the “Known_y’s” (the dependent variable) & the absorbance data as the “Known_x’s” (the independent variable).  The data displayed in the 2x5 box will give you the slope & y-intercept of the linear relationship of protein mass vs. absorbance.  Use this approximated linear relationship to calculate the protein mass present in your unknowns from their measured absorbances directly- so you do not need to know the volume, extinction coefficient, or the length of the cuvette (as long as they are all constant) to estimate the protein mass present in the unknowns from absorbances.  Divide the mass of protein present in an unknown sample by the volume of that unknown that you added to the reaction tube to determine the concentration, by mass, of protein in the unknown.

by Mufasa

Edited by Justin Maresh