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 – Removal of cell debris by centrifugation • 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 Affinity Chromatography: the General Strategy Specific binding Example: Elution (release) Can use altered pH, salt, competitor molecule for elution. 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). Imidazole & histidine: a structural comparison Imidazole ring • Excess imidazole outcompetes 6xHis-b-gal for binding to Ni+ agarose b-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) Fractioncontains b-galactosidase-PF Determining Total Protein Content Spectrophotometrically (both CE and PF) Free Coomassie Blue Dye (Bradford Reagent) Absorbance at 470 nm + Protein Dye Bound to Protein Absorbance at 595 nm Absorbance Making a Standard Curve from Absorbance readings of known BSA concentrations using Linear Regression Concentration (mg/ml) • y =m x + b, where m is the slope & b is the yintercept. 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) Measuring the Specific Activity of b-gal Colorless • 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. 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. Calculating the Concentration of ONP from A420 Readings of Enzyme Reactions Beer-Lambert Law: A C= e*l 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) e for ONP = 4800 M-1cm-1 • 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 (0.11.0). Will try several dilutions of CE & PF. Making Dilutions V1 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.) 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 b-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. 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.