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Bio-Rad Protein Assay (Bradford) The principle of the Bradford assay is the shift of the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 from 465nm to 595nm when binding to proteins occurs. Samples are measured with the HP8452A spectrophotometer using an automated analysis method. 1. Calibration - Place 0µl, 5µl, 10µl, 15µl, 20µl, 25µl, 30µl (0-30µg) of a 1mg/ml stock solution of BGG (or BSA) in a microfuge tube,

add H2O to 800µl. - Add 200µl Dye Reagent Concentrate and vortex. Transfer to 1ml plastic cuvette and wait 15min before starting the

measurement. - Turn on HP8452A and computer (Pwd = "thch") 10min before measuring so that the lamp light is stable. - Open the HP8452A software and select the Advanced Quant module. - Load parameter file: Files > Recall Parameter > BRADY.QSP - Manually select an integration time of 5s: Parameter > Instrumental > Integration time 5.0s - Delete old standards: Edit > Delete Standards > All - Move transport to position 1: Parameter > Multicell Transport > Position > 1 - Measure blank (H2O): Measure > Blank - Measure standards: Measure > Standard [Specify Name: BGG or BSA, Concentration: 0-30, Units: µg] - When all standards have been measured make calibration: Task > Calibrate - Before saving, check quality of calibration curve: Graphics > Calibration Curve > Calibration Curve Calculation 1 - When calibration is ok save it: Files > Store Calibration [Specify Filename: BRADYBGG or BRADYBSA] - When asked Override File BRADYBGG.QCL or BRADYBSA.QCL say OK - Print new calibration and place copy in Protocols Folder: Hardcopy > Print Calibration 2. Samples - Place sample containing up to 30µg of protein in a microfuge tube and add H2O to 800µl. - In each series, include controls without protein (only H2O) and with 10µg BGG or BSA. - Add 200µl Dye Reagent Concentrate and vortex. Transfer to 1ml plastic cuvette and determine protein concentration

15-30min after addition of reagent using the automated analysis method: - Turn on HP8452A and computer (Pwd = "thch") 10min before measuring so that the lamp light is stable. - Open the HP8452A software and select the Advanced Quant module. - Load parameter file: Files > Recall Parameter > BRADY.QSP - Manually select an integration time of 5s: Parameter > Instrumental > Integration time 5.0s - Place a cuvette with H2O in position 1 and your samples in positions 2-7 of the multicell transport. - Start measurement: Files > Run Method > BRADY.QAU (Results are stored in BRADY.QRS) - To view results on the screen: Task > Analyze > Display - To print results: Hardcopy > Report > BRADY.QRS 3. Important considerations - Due to its high viscosity, the Dye Reagent Concentrate should be pipeted very slowly to transfer the correct volume. - If the controls are more than 2µg off their expected value, repeat the assays including additional controls. If these are also

off the expected values, make a new calibration. - The most accurate measurements can be made in the range of 5-25µg, adjust sample volume accordingly. - For substances interfering with the Bradford method consult the Biorad Protein Assay Kit manual. 4. Reagents - Dye Reagent Concentrate: Biorad Protein Assay Kit I, store at 4°C - BGG: bovine gamma globulin, dissolved in H2O at 1mg/ml, store at -20°C - BSA: bovine serum albumin, dissolved in H2O at 1mg/ml, store at -20°C CONTRIBUTED BY: Hubert Schwelberger (hubert.schwelberger@i-med.ac.at) LAST MODIFIED: 2012-06-21