User:Sayed Massie Husaini/Notebook/september 5 2017/2017/09/19

Project name

Main project page Previous entry      Next entry

Bradford Analysis Prepared 50 mL of a stock solution of BSA that is roughly 5 mg in 5 mL of saline. Used a quartz cuvette to record UV-VIS spectra between 200 nm and 800 nm. Recorded UV-VIS spectrum for saline. Make 6 - 8 standard solutions (1 mL each) between 1 μg/mL and 20 μg/m using a serial dilution. Determined the appropriate volume of stock solution to use and add it to a 1.5 mL centrifuge tube. 200 μL was added of the Bio-Rad Protein Assay reagent using 1:4 concentrate diluted with water. Added the correct amount of Tris/NaCl buffer such that the final volume is 1 mL. Closed the tubes and vortex them for 5 - 10 sec. Let them sit for 5 min. Obtained a UV-VIS spectrum. used PS cuvettes. recorded between 400 nm and 800 nm Make duplicate blanks (4 solutions total) as well 1 mL Tris/NaCl buffer 200 μL Bradford reagent + 800 μL buffer recorded their UV-VIS spectra between 400 nm and 800 nm Bradford Analysis in Use: Diluted the BSA and original AA stock solutions to the appropriate range. Used Bradford Analysis to determine the concentration. Heated BSA solution using a block heater to denature (unfold) the protein. Place 1 - 2 mL of solution in microcentrifuge and place in 95°C heating block for 5 min. Tried adding some SDS or guanidinium chloride Prepare 10 mL of 2% SDS and 10 mL 2% GuanCl Add equal mixtures of SDS and protein. Use Bradford analysis. Add equal mixtures of GuanCl and Protein. Use Bradford Analysis. Calculations for Bradford: