530L_notes_lecture7-2

530L_notes_lecture7-2 - 1. We use the protein standard BSA...

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IV. Elution profile (see ppt slide 1) – Where is our protein Intro to Double Y axis 1. total protein; total acivity vs. elution volume (fraction number) 2. [NaCl] is also indicated (load, wash, and gradient phases) Fractions collected 1. Each contains different amounts of different proteins 2. How do we determine which one has our protein? Methods: 1. Total protein: Bradford Assay – to determine protein concentration 2. Enzyme activity assay: PNPP PNP using spectrophotography at 410 nm A. Bradford Assay: Purpose is to determine the concentration of all the protein in a particular fraction. “over-expressing the protein” What will this tell us? B. Bradford theory: 1. Dye- coommassie blue G-250. What does it do? In the absence of proteins, color and Amax: redish brown 470 nm
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In the presence of bound proteins, color and Amax: blue 595 nm Important note: intensity of color! More intense, more concentration qualitative analysis Generation of a standard curve for a Bradford assay
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Unformatted text preview: 1. We use the protein standard BSA Why do we use BSA? Cheat as dirt Average amino acid composition standard protein 2. Method: making a calibration curve (see ppt slide 2) Known concentration of BSA to brafford reagent.s 3. after making the std curve, you need to assay YOUR fractions and determine [protein] Look at ppt slide 1 Fraction 7 = 0.15 mg/ml Total protein = [protein] mg/ml X vol of fraction (ml) = total protein in mg = 0.15 mg/ml X 1.5 ml = 0.225 mg 4. Enzyme activity (our second assay) Purpose: determine alk phos activity in our fractions (the same ones we used for our Bradford assay) How much activity is there in this fraction tube? See ppt slide 2. The nuts and bolts of the activity assay: Determining total activity A410 /3 (min)/vol assayed (ml) X total vol of fraction (mls) = A410/min (convert A410 to concentration using Beers law)...
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530L_notes_lecture7-2 - 1. We use the protein standard BSA...

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