User:Susan Schultz/Notebook/Experimental Biological Chemistry/2011/09/13

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MBP Concentration Determination Main project page
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Objective

Use a series of BSA standard solution to determine the linear relationship between protein concentration and absorbance.

Use the regression line to determine the concentration of an unknown sample of Maltose Binding Protein.

Prepare a starter culture for bacterial growth and protein expression.

Description

A calibration curve was established using six standard solutions of BSA. The standard solutions of approximately 0.73, 1.46, 3.65, 5.84, 8.03, and 10.22μg/mL were prepared and then added to 200 μL of Bio-Rad protein assay. In disposable cuvettes a UV-VIS spectra was taken from 200-800nm using a Shimadzu UV-2550 UV-VIS spectrophotometer. Specifically the absorbance of each solution at 595nm was noted. A regression line was found to fit the absorbance vs. concentration data. In the future this correlation may be used to establish the concentration of MPB in a solution. This method was then tested to determine the concentration of an unknown sample of MPB. A 1/1000th dilution of the unknown sample was added to 200 μL of Bio-Rad protein assay. The measured absorbance was used with regression line established from the BSA standards to determine the concentration. A reference spectra of BSA in water was also taken using a quartz cuvette.

Image:BSA Lines.jpg


A rich broth was prepared using 10 g tryptone, 5 g yeast extract, 5 g NaCl, 2 g glucose per liter of distilled water. Four starter culture mediums were prepared in 250mL flasks with 25mLs of rich broth medium. They were covered in foil and autoclaved. An autoclaved micropipette tube was then used to transfer a sample of R2566 E. coli with the pMAL-pIII plasmid into each flask. The pipette tip was touched to the bacteria and then dropped in the flask. 100μg/mL of ampicillin and was added to each flask, and then they were placed in a shaker/incubator. They were shaken at 250rpm at 37°C overnight.

Data

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Figure 1. Absorbance of MBP standards

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Figure 2. The absorbance of protein in water adjusted so that A→0.

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Figure 3. Molar Absorbtivity v. Wavelength for protein in water


The asjusted absorbance of the unknown MBP at 595nm was 0.015. Using the regression line from the BSA standards the concentration of the MBP in the sample was 19.02nM. The sample was 1,000X diulte, so the unknown sample was 19.02μM.

Notes

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