BSA Assay: Accurate Protein Quantification Using Bradford Method

Lab Progress Report #1 BSA Assay Alejandra Quintana Team: Gossypol 02/05/2024

Introduction Quantifying proteins is a part of research as it offers valuable insights, into how cells work the causes of diseases and the creation of potential treatments. Bovine Serum Albumin (BSA) is commonly used as a protein to serve as a reference in protein quantification assays because of its well known structure and stability (Bradford, 1976). For this experiment our goal was to measure the amount of BSA in a given sample using the Bradford Assay, which's an used and trustworthy method based on spectrophotometry. The principle, behind the Bradford Assay is that when Coomassie Brilliant Blue G 250 binds to proteins it undergoes a color change that can be detected at a wavelength (Bradford, 1976). By measuring the degree of color change we can accurately determine the concentration of proteins, in the sample ( Friendenauer , 1989) . The goals of this experiment involve creating a reference curve using known amounts of BSA identifying the range, in which the assay's linear and using the method to measure the concentration of a sample (Zor, 1996). Furthermore, we will evaluate the precision and accuracy of the assay to determine its dependability, for regular protein quantification. It is crucial to have an understanding of the levels of BSA in biological samples for many research purposes. These include protein purification, enzyme kinetics and drug development. Precise measurement and quantification of BSA are essential, for standardizing outcomes and maintaining the reliability and reproducibility of studies. This experiment aims to improve and optimize protein quantification techniques by performing the Bradford Assay. The goal is to enhance the accuracy and dependability of analyses in scientific fields.

Procedure To get ready, for the protein quantification test, there were eight tubes collected to make dilutions. Each tube was carefully labeled with numbers from 1 to 8. Tube 1 was first filled with 450 μl of phosphate buffered saline (PBS) and tubes 2 to 8 received 300 μl of PBS each. After that 150 μl of the BSA solution was added into tube 1, and it was resuspended. To start the dilution process 300 μl of the solution was transferred, from tube 1 to tube 2. This step was repeated for each tube going from tube 2 to tube 3 then, from tube 3 to tube 4, and so on until tube 7 was reached. Tube 8 which is designated as the blank was kept with a volume of 300 μl of PBS. Moving on, using a 96-well plate the plate was labeled with the team name and the tube numbers that matched the samples. In order to make everything consistent 100 μl of working reagent were carefully pipetted into all the wells that were meant for dilutions. Additionally, four wells were allocated, for the unknown protein sample. In the dilution series, 100 μl of each dilution was carefully pipetted into wells. A bottom- to-top approach was used, within the plate starting from the concentration (tube 8) and moving up to the concentration (tube 1). At the time we also pipetted 100 μl of the unknown protein sample into the four extra wells. After adding the sample and reagents the plate was left at room temperature for 30 minutes to allow the reaction and binding process to occur. Then the absorbance readings were measured at 562 nm to determine the protein concentrations in the samples that were prepared.

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

Results and Data Analysis 0 100 200 300 400 500 600 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 f(x) = 0 x + 0.11 R² = 0.78 Standard Curve Concentration (ug/ml) Absorbance The absorbance readings obtained from the BSA experiment were analyzed at concentrations ranging from 500, 250 125 62.5, 31.25, 15.625, 7.8125 and 0 μg/ml. It

was observed that the average absorbances corresponding to these concentrations were as follows; 0.5355, 0.3415, 0.407, 0.218, 0.148, 0.118, 0.048, and -0.0065 respectively. Enough there is a trend, in the absorbance values which indicates a decrease in proportion as the BSA concentration diminishes. However, it is crucial to address the R² which resulted in 0.7755. Conclusion To sum up the purpose of this experiment was to measure protein concentrations using the Bradford Assay, which's an used method that relies on spectrophotometry. The experiment consisted of making BSA dilutions creating a curve and assessing the precision and accuracy of the assay. The basic principles of the Bradford Assay were used to measure the concentrations of BSA at levels of dilution. The absorbance readings showed a pattern indicating that as the BSA concentration decreased there was a decrease, in absorbance. However it is important to acknowledge the absorbance observed at the lowest concentration, which suggests the presence of experimental noise or procedural inconsistencies that need to be investigated further. The R² value, which stands at 0.7755 highlights the assays reliability under the conditions. This suggests a connection, between the known BSA concentrations and the recorded absorbances. These findings, combined with the observed trend offer insights into how dependable and accurate the Bradford Assay is, for protein quantification. While this experiment came very close to the wanted R² value, which is 1, exploring potential modifications to improve accuracy, for example, pipetting techniques. References

1. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 , 248–254. 2. Friendenauer, S. and Berlet, H.H. (1989) Sensitivity and variability of the Bradford protein assay in the presence of detergents. Anal. Biochem. 178 , 263– 268. 3. Zor, T. and Selinger, Z. (1996) Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal. Biochem. 236 , 302–308.

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version

Access to all documents
Unlimited textbook solutions
24/7 expert homework help

Lab Progress Report #1

Related Documents