spectrophotometry

Lab 4. Spectrophotometry Introduction: The purpose of this lab experiment is the determination of protein concentration in a solution. From the variety of used methods to determine protein concentration you will use in our experiment the Bradford Protein Assay. It consists in the development of color by Coomasie Blue in presence of proteins. The developed complex will absorb light at 595 nm wavelength of light. The color development is proportional to the protein concentration. Since concentration and absorbance are proportional the application of the Beer’s Law makes possible to draw a standard curve by plotting absorbance vs. concentration. Absorbance values will be measure in a spectrophotometer Materials and reagents: 1 Spectrophotometer and 1 cuvette 1 vortex 5mL pipette for Bradford. 250 mL Erlenmeyer to serve as pipette holder 1 pipette filler. Pipette stand with 4 pipettes (P10, P100, P100, and P1000) Pipette tips for all pipettes 8 test tubes Deionized water 0.5 mL of bovine serum albumin (BSA) 25 mL of Bradford Reagent (filtered) Unknown protein solution A Unknown protein solution B Procedure: Warm the spectrophotometer for no less than 15 minutes. Select the wavelength at 595nm. In six test tubes add Bovine Serum Albumin according to the amount specified in the table. Reagent/Tube (?L) Unknowns 1 2 3 4 5 6 A B BSA Standard 1 mg/mL 0 10 20 30 40 50 0 0 Unknown protein 0 0 0 0 0 0 30 30 Bradford reagent 3.0 mL _____________________________________________________ Zero the spectrophotometer with the first tube that contains only Bradford reagent but not protein. Add 3.0 mL Bradford reagent to the second tube. Vortex the mixture for 15 seconds. Let the mixture set for 10 minutes. Read and record the absorbance. Repeat the same procedure to all other tubes and the unknown samples Results: Draw the table you obtained. 812576000 Graph your standard curve by plotting protein content in the abscissa and absorbance in the ordinate. Calculate the protein content of your samples. BSA Volume Protein concentration 0 0 10 12 20 24 30 34 40 43 50 49 Unknown 110 Results table Bradford (mL) BSA (?L) Absorbance 3 0 0 3 10 0.207 3 20 0.391 3 30 0.542 3 40 0.681 3 50 0.789 3 30 1.751 3 30 Conclusions: This lab was about determining the concentration of protein in a solution. The procedure that was used to determine the concentration of proteins was the Bradford protein essay and the spectrophotometer to measure the absorbance. A graph was set up and a slope was determined. The concentration of the protein was obtained by using the formula of (the inverse of the slope multiplied by the absorbance).There was many sources of error in this experiment mainly human errors such as pipetting, timing and measurements techniques. Also when using a spectrometer and just one cuvette for all the samples, the cuvette might have gotten stained, therefore it could give wrong readings.