Presentation on theme: "BISC 220 Lab 2 Protein Purification by Affinity Chromatography & Determination of Specific Activity."— Presentation transcript:
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 -gal in each. Quantify & compare total protein & specific activity for both fractions & then calculate % yield & purification factor
Affinity Chromatography: the General Strategy Example: Specific binding Elution (release) Can use altered pH, salt, competitor molecule for elution.
Protein Purification via Metal Chelate Affinity Chromatography The 6xHis tag on the -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 -gal will be eluted (released) from the beads by competition with imidazole (a molecule similar to histidine).
Imidazole ring Imidazole & histidine: a structural comparison Excess imidazole out- competes 6xHis- -gal for binding to Ni+ agarose
-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
Determining Total Protein Content Spectrophotometrically (both CE and PF) Free Coomassie Blue Dye (Bradford Reagent) Absorbance at 470 nm Dye Bound to Protein Absorbance at 595 nm + Protein
Making a Standard Curve from Absorbance readings of known BSA concentrations using Linear Regression 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) Concentration (mg/ml) Absorbance
Measuring the Specific Activity of gal Colorless ONPG = artificial substrate for -gal Specific activity = V max (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.
Beer-Lambert Law:C = A * l C = concentration (moles/L) A = absorbance reading at given wavelength (no units) = molar extinction coefficient at given (M -1 cm -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. for ONP = 4800 M -1 cm -1 Calculating the Concentration of ONP from A 420 Readings of Enzyme Reactions
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 -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.