Protein Concentration lab writeup

Protein Concentration lab writeup - Using Spectrophotometry...

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Using Spectrophotometry to Understand Cellular Proteins Amber Elkins Dr. Bagavandoss October 05, 2007
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Introduction: The purpose of this experiment was to learn how to use the spectrophotometer to establish a standard curve for bovine serum albumin (BSA). Subsequently, the curve will be used to determine the concentration of a mixture of proteins in an unknown sample prepared from wheat germ to later determine the activity of an enzyme in the extract. Materials & Methods: Supplies needed included: 1 spectrophotometer Balance and weigh boats 1.5 mL microtubes 8 15 mL conical tubes 4 50 mL orange cap tubes 4 50 mL beakers Bradford reagent BSA protein standard (2 mg/mL) White racks and donuts Box of 12 X 75 disposable glass test tubes Cuvettes Vortex Acetate buffer (0.1 M), pH 4.5 2
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Extraction Bugger/diluent 1: 0.1 M acetate buffer with Triton X-100 (0.05%), pH 4.5 which will facilitate the lysis of the wheat germ cells (kept on ice) (500 mL) Fresh wheat germ First, the wheat germ was prepared by weighing out about 0.5 grams of wheat germ (0.501 g was used) and placed into a homogenizer. Five milliliters of cold extraction buffer was added to the wheat germ. The tissue was homogenized with a pestle until a suspension was formed and transferred into a 15 mL conical centrifuge tube. The tube was counter balanced with another 15 mL tube with water and centrifuged for 10 minutes at 3400 RPM. Another
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Protein Concentration lab writeup - Using Spectrophotometry...

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