Lab 2 Protein Determination BIO462 - Biochemistry PDF

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Experiment 2: Protein DeterminationPlease watch the video and answer all questions.youtu/hdb3s4YHkms youtu/iNmmkZXgbf Name the dye used in Bradford Assay (1 mark). Coomassie Brilliant Blue State the colour change that occurs when proteins combine with the dye reagent (1 mark). Change colour from bro...

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Experiment 2: Protein Determination

Please watch the video and answer all questions. https://youtu.be/hdb3s4YHkms https://youtu.be/iNmmkZXgbf0 1. Name the dye used in Bradford Assay (1 mark). Coomassie Brilliant Blue 2. State the colour change that occurs when proteins combine with the dye reagent (1 mark). Change colour from brownish red to blue. 3. In Lowry protein assay, name the bond in protein that binds to copper ions (1 mark). Peptide bonds 4. A Bradford assay was conducted to determine the total protein concentration in a sample. A volume of 2 μL of the original sample was diluted to 100 μL with a buffer before performing the assay. The diluted sample gave an absorbance at 595 nm of 0.255. Using the data for the standards below, Protein concentration (μg/mL) 25 125 250 500 750 1000 1500

A595

0.008 0.087 0.113 0.197 0.295 0.429 0.608

a) Plot a standard curve based on the data given (3 marks).

Measurement of the Protein Concentration 0.7 y = 0.0004x + 0.0117

0.6

Absorbance (595nm)

0.5 0.4 0.3 0.2 0.1 0 0

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Protein concentration (ug/mL)

b) Determine the concentration of protein in the diluted sample (2 marks). Absorbance = 0.255 Y = 0.0004x + 0.0117 X = (0.255-0.0117)/ 0.0004 = 608.25 ug/mL

c) Calculate the total protein concentration in the original sample (2 marks). M1V1 = M2V2 M1(2uL) = (100uL)(608.25ug/mL) M1 = 30412.5 ug/mL

1600...