Bradford Assay And How To Calculate Protein Concentration In Excel

This video demonstrates how to perform a Bradford dye assay for the determination of protein concentrations. You will need a kit including Bradford reagent and bovine serum albumin (BSA) protein standards. This particular assay uses a 96-well plate and platereader to measure absorbance at 595 nm, but the assay can also be done using a single cuvette spectrophotometer. The protein concentration can be calculated by graphing a standard curve in Microsoft Excel then using a non-linear fit and the Goal Seek function. The steps to this protocol are: 1) Read the instructions that came with your Bradford kit. 2) Dilute BSA protein standards two fold to create a dilution series between 2 - 0.125 mg/mL. Include a 0 mg/mL (buffer only) control. 3) Mix a little bit of the unknown protein with Bradford Reagent and observe the blue color. If it is pure blue, dilute part of your sample until mixing it with Bradford dye results in a blue / red mixture. (similar to 0.5 mg/mL BSA protein standard when mixed with the dye). 4) Add 10 uL of BSA standard solutions to a 96-well plate. 5) Add 10 uL of the unknown protein sample to the 96-well plate. 6) Add 200 uL of Bradford dye reagent to each well and mix for 30 seconds. 7) Wait 10 minutes then read the absorbance of each well at 595 nm using a platereader. 8) Export the data into Excel and graph the standards. Fit to a polynomial line and display the equation for the line. 9) Use the equation and Excel's Goal Seek function to find the concentration of your unknown protein samples by comparison to the standard curve. Here is a link to the Excel sheet created for this video: https://docs.google.com/spreadsheets/... We hope you enjoyed this video. If you are interested in Solidzymes' protein purification, enzyme activity optimization, or enzyme immobilization services please visit our website: https://solidzymes.com/