Jonathan Nguyen Megan Ringling MCB253 09 February 2024 Background The purpose of this experiment is to determine the concentration of the unknown BSA protein We will be utilizing the concept of protein dye binding in a Bradford Assay to carry out the experiment The Bradford assay technique is based on the observation that Coomassie Brilliant Blue G 250 exists in two different color forms red and blue The red form is converted to the blue form upon binding of the dye to protein The protein dye complex has a high extinction coefficient thus leading to great sensitivity in measurement of the protein The binding of the dye to protein is a very rapid process approximately 2 min thus making the procedure very rapid Bradford 1976 p 249 The Bradford assay s ability to measure protein concentration is based on the shift in absorbance maximum of Coomassie Brilliant Blue G 250 dye from 465 to 595 nm following binding to proteins in solution Hypothesis If I produce a standard curve of concentration vs absorbance with an R 2 value greater than 0 95 then the predicted concentration of the unknown BSA protein 1 that we calculate should be accurate Procedure 1 Make 50 l of each BSA standard as shown in Table 1 2 1 2 mL of Bradford dye coomassie blue into each cuvette using the P1000 pipette 3 Add 24 L of your BSA standard or blank PBS using P200 pipette 4 Mix thoroughly with pipette 5 Wait for 10 min at room temperature 6 Read absorbance of each cuvette in spectrophotometer at 595 nm 7 Create a graph of BSA concentration vs absorbance a Make sure to show graph equation and R squared value 8 Calculate concentration of unknown samples using graph equation 9 Repeat steps 2 6 for the 7 unknown dilution samples a 100 L PBS 0 L unknown BSA b 1 fold 50 L PBS 100 L unknown BSA c 2 fold 50 L PBS 50 L of 1 fold dilution d 4 fold 50 L PBS 50 L of 2 fold dilution e 8 fold 50 L PBS 50 L of 4 fold dilution f 16 fold 50 L PBS 50 L of 8 fold dilution g 32 fold 50 L PBS 50 L of 16 fold dilution Data Dilution Charts Table 1 BSA Standard mg mL PBS Buffer L Stock BSA L 2 mg mL Absorbance 595 nm Concentration mg mL 0 0 25 0 50 0 75 1 0 1 25 1 50 Table 2 0 1 fold 2 fold 4 fold 8 fold 16 fold 32 fold 50 43 75 37 5 31 25 25 18 75 12 5 100 50 50 50 50 50 50 0 6 25 12 5 18 75 25 31 25 37 5 1 L 0 100 50 unknown 50 of 2 fold 50 of 4 fold 50 of 8 fold 50 of 16 fold Serial Dilutions PBS Buffer L Unknown BSA Absorbance 595 nm Concentration mg mL Insert standard graph here Explanation of Expected Results After producing our BSA standard samples we expect an overall increase in concentration and absorbance correlated with an increase in BSA standard concentration We expect this since concentration is directly related to absorbance and increasing the concentration of BSA protein would increase the absorbance of the solution and consequently the concentration After producing our BSA unknown samples we expect an overall decrease in concentration and absorbance after each subsequent dilution since we will be adding a smaller amount of unknown BSA while maintaining the same volume of PBS buffer 50 L for each serial dilution After adding our protein the Coomassie Blue dye should change color from reddish to blue which indicates the presence of protein We should produce a standard curve graph of concentration vs absorbance of BSA that has a R 2 value greater than 0 95 because this would allow us to accurately calculate the concentration of unknown protein 1 Some possible sources of error that may affect the accuracy of our measurement of protein absorbance include the loading time of our cuvettes in the spectrophotometer impurities in the cuvette cuvette material and dye temperature References Bradford M 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding Analytical Biochemistry 72 1 2 248 254 https doi org 10 1006 abio 1976 9999