Bradford Assay Matt Shipman 2006 Adapted and modified from a protocol by Britt Alsaker.

Purpose: To determine the concentration of protein in a sample. Preparation: 1) Prepare/label 1.5 mL Eppendorf tubes for curve and sample replicates. You will need 3x7 tubes for the standard curve, and 5 tubes per sample. Pour out the approximate amount of Bradford reagent (obtainable from Biorad) that will be required into a clean beaker and cover it with Parafilm. It is better to overshoot a little than undershoot on the volume of Bradford reagent. Set the Bradford reagent aside and allow it to come to room temperature before using it. 2) Add nanopure water to the tubes as indicated in the table below. Add protein as indicated in the table below. Note that the standard curve is calculated on a BSA standard (Biorad Quickstart) at a concentration 2 mg/mL. Adjust accordingly for other concentrations. Proteins other than BSA may be used at the operators discretion, however it is recommended that a commercial standard be used for improved accuracy and a more consistent batch. For the sample replicates more protein may be used for dilute samples, but the volume of water must be adjusted accordingly so that the total for vol. of water + vol. of sample = 800 uL. 3) Add the Bradford reagent as indicated below. Vortex the samples well and set them aside for 15 minutes to allow the samples to equilibrate. The total volume of each sample is 1 mL. 4) Pipette 200 uL of each sample into a clear 96-well plate. Use a different pipette tip for each tube. Scan on a plate reader at 595 nm. Data analysis: 5) Prepare an Excel (or other) spreadsheet as indicated in the template below. Graph the average column for the A595 values in a scatter plot. Add a trendline and display both the equation for the line and the R2 value for the curve. A five-point calibration curve is all that is required for this test. The remaining two points can be discarded at the operators discretion. The R2 value for the curve should be 0.995 or better. It is recommended that the trendline fit to the curve be a linear fit. Other curve fits are allowed so long as it is possible to solve for X. 6) Solve the equation generated by the curve for x. X is the concentration of the protein in the sample. Note the Y-intercept and the slope of the line.

7) Calculate the protein concentrations in the sample replicates as follows: [(Sample A595-Y intercept)/(slope of the line)]/(volume of protein added to each sample) 8) For each set of sample replicates determine the average value of the set. Up to two points may be removed from each set of five replicates provided that they are >10% different from the mean. Points <10% different from the mean may not be removed for any reason. Note that three replicates is the minimum required for each sample average, if this cannot be satisfactorily achieved, the assay should be run again. Also compute the standard deviation. 9) Samples (or standard curves) displaying a high degree of variation should be repeated. The replicates (curves and samples) should be sufficient to account for pipetting error inherent in the pipette(s) being used, but will not account for significant operator error. Notes: 10) It is allowed for the operator to filter each tube with a Whatman #1 filter paper in between steps 3 and 4 above to remove excess Bradford reagent. This is not necessary (so long as all tubes are treated the same) and will only change the location of the Yintercept in your curve. 11) It is highly recommended that the standard curve and the protein replicates be pipetted with a P20 using a P200 pipette tip. Smaller pipettes cannot handle the P200 tips, and smaller tips may actually increase pipetting errors. The smaller diameter of the smaller tips are more likely to cause the sample to wick up into the tip due to surface tension. Templates:
Sample BSA standard 1 BSA standard 2 BSA standard 3 BSA standard 4 BSA standard 5 BSA standard 6 BSA standard 7 Sample Sample Sample Sample Sample replicate 1 replicate 2 replicate 3 replicate 4 replicate 5 uL ddH2O 800 799.5 799 797.5 795 790 787.5 799 799 799 799 799 uL sample or BSA (2 mg/mL) 0 0.5 1 2.5 5 10 12.5 1 1 1 1 1 uL Bradford reagent 200 200 200 200 200 200 200 200 200 200 200 200

Curve BSA standard 1 BSA standard 2 BSA standard 3 BSA standard 4 BSA standard 5 BSA standard 6 BSA standard 7 Sample replicate 1 replicate 2 replicate 3 replicate 4 replicate 5

A595 curve 1

A595 curve 2

A595 curve 3

Average A595 for all curves

A595

[Protein] Average Standard Deviation

Sample Sample Sample Sample Sample