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BISC 220 Lab 2

BISC 220 Lab 2. Protein Purification by Affinity Chromatography & Determination of Specific Activity. TO DO TODAY. Extract protein induced last week from the bacterial cells chemical cell lysis with“Bacterial Protein Extraction Reagent”- DETERGENT

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BISC 220 Lab 2

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  1. BISC 220 Lab 2 Protein Purification by Affinity Chromatography & Determination of Specific Activity

  2. TO DO TODAY • Extract protein induced last week from the bacterial cells • chemical cell lysis with“Bacterial Protein Extraction Reagent”- DETERGENT • Removal of cell debris bycentrifugation • Partial Purification of protein • Separate the protein of interest from other proteins by Affinity Chromatography • Assay the total protein in the CRUDE EXTRACT & the PURIFIED FRACTION and Assay the specific activity of the b-gal in each. • Quantify & compare total protein & specific activity for both fractions & then calculate % yield & purification factor

  3. Affinity Chromatography: the General Strategy Specific binding Elution (release) Can use altered pH, salt, competitor molecule for elution. Example:

  4. Protein Purification via Metal Chelate Affinity Chromatography • The 6xHis tag on the b-gal will bind tightly to the Ni+ agarose, which can be separated from the supernatant by centrifugation. • Other proteins that interact non-specifically (weakly) with the Ni+ agarose will be removed during washes. • The 6xHis b-gal will be eluted (released) from the beads by competition with imidazole (a molecule similar to histidine).

  5. Imidazole & histidine: a structural comparison Imidazole ring • Excess imidazole out- competes 6xHis-b-gal for binding to Ni+ agarose

  6. -Gal Partial Purification Protocol:Things to Remember • Follow directions carefully • Know where you are in process • Mix well, measure carefully & don’t confuse reagents • Be careful making dilutions • End up with 2 fractions to assay: • Crude Extract-CE • Purified (partially) Fraction- contains -galactosidase-PF

  7. Free Coomassie Blue Dye (Bradford Reagent) Absorbance at 470 nm Dye Bound to Protein Absorbance at 595 nm Determining Total Protein Content Spectrophotometrically(both CE and PF) + Protein

  8. Concentration (mg/ml) Making a Standard Curve from Absorbance readings of known BSA concentrations using Linear Regression Absorbance • y =m x + b, where m is the slope & b is the y- intercept. Use the equation generated by Excel to solve for x (concentration) • Will need to dilute a 1 mg/ml BSA stock to make 0.1, 0.2, 0.4, 0.6 & 0.8 mg/ml samples (not a dilution series; make 200 µl of each dilution)

  9. Colorless Measuring the Specific Activity of b-gal • ONPG = artificial substrate for b-gal • Specific activity = Vmax (maximum velocity) = rate of appearance of product under conditions of saturating amounts of substrate (mmol/min/mg protein) • Detect appearance of ONP (product) by absorbance at 420 nm.

  10. What does the Specific Activity tell you? • With greater purification of an enzyme, total activity & % yield will decrease but specific activity will increase. • Purification factor = ratio of specific activities of PF/CE; a higher purification factor means that more of the protein in the sample is the enzyme of interest.

  11. A Beer-Lambert Law: C = e * l • e for ONP = 4800 M-1cm-1 Calculating the Concentration of ONP from A420 Readings of Enzyme Reactions • C = concentration (moles/L) • A = absorbance reading at given wavelength (no • units) • e = molar extinction coefficient at given l • (M-1cm-1) • l = spectrophotometer path length (cm) • Must use an amount of enzyme that produces an amount of product (in a defined reaction time) that gives an absorbance reading in the reliable range for the spectrophotometer (0.1- 1.0). Will try several dilutions of CE & PF.

  12. Making DilutionsV1 x C1=V2 x C2 • FOR PROTEIN ASSAY 1. BSA stock 1mg/ml Working dilutions (want 200µl): 0.1, 0.2, 0.4, 0.6, 0.8 mg/ml 2. Purified (PF) & Crude Extract (CE) 1:5 dilution with Z buffer (want 300 µl) • FOR ENZYME ASSAY 1. Purified Fraction Want 250µl each of 1:100, 1:200, 1:400& 1:800 dilutions 2. Crude Extract Want 250 µl each of 1:50, 1:100, 1:200 & 1:400 dilutions (Use serial dilution strategy for these.)

  13. Things to Remember about Assays: Protein Assay • Make 2 reagent blanks instead of 1 since using double beam spectrophotometer- 11 tubes • Mix dilutions well & keep on ice • Timing is not critical -Gal Assay • Keep all diluted fractions on ice • Make 2 reagent blanks instead 1 • Timing IS critical!!! • You will have a lot of tubes in your ice bucket. Be VERY careful not to mix things up—label well.

  14. Before You Leave • Add glycerol to remaining purified fraction & give to instructor to freeze • Give 3 samples to instructor (properly labeled—see p. 41) • Clean up your work area Homework • Complete calculations & answer questions on p. 42 • Don’t wait until the last minute to do the calculations! • When calculating protein concentrations, don’t forget to account for the dilutions. • For other calculations, follow examples on p. 48-50.

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