1 Purification and Characterization of T7 RNA Polymerase Experiment 2 of 2 CHEM 4581: Biochemistry Laboratory I Version: February 16, 2008 BACKGROUND AND GOAL In Part 2 of this two-week study, students will characterize the fractions obtained from the purification of T7 RNA Polymerase from E. coli. The fractions will be analyzed for protein content using the Bradford Protein Assay and for purity and MW determination using SDS-PAGE. MATERIALS AND REAGENTS Bradford Protein Assay Reagent (Bio-Rad) Bovine Serum Albumin (BSA; Pierce) Disposable Cuvettes with 2 optical windows – transparency in visible region Shimadzu UV-1601 Spectrophotometer Mini-Protean 3 Vertical Gel Electrophoresis System (Bio-Rad) Power Supply Rocking Platform High MW Marker, Bio-Rad 1.5 M Tris-HCl, pH 8.8 0.5 M Tris-HCl, pH 6.8 10% (w/v) SDS 40% Acrylamide:Bisacrylamide Solution (37.5:1 molar ratio) 10% (w/v) Ammonium Persulfate – prepare fresh daily N,N, N’,N’-Tetramethylethylenediamine (TEMED) Laemmli Sample Buffer – must supplement with 2-mercaptoethanol just before use 10X Electrophoresis Buffer Coomassie Stain Solution Destain Solution EXPERIMENTAL PROCEDURES Determination of Protein Concentration 1. Prepare a 4-fold dilution of a 2 mg/mL BSA sample by adding 50 μL of 2 mg/mL BSA to 150 μL of dI water to make 200 μL of 0.5 mg/mL BSA. This working solution is what you will use from then on in this experiment. In general, protein solutions should be kept on ice, but this experiment will totally destroy the protein anyway, so room temperature storage is sufficient over the course of the experiment. 2. Generate test samples for the reference cell, blank, BSA standards and any protein sample to be tested according to Table 1 in disposable cuvettes. Combine protein sample and dI water in each labeled cuvette first. Then, systematically add Bradford Protein Assay Reagent to each tube in the order in which the sample’s absorbance will be measured.2 Table 1. Preparation of Samples for Bradford Protein Assay *Note: Sometimes 2.5 μg/mL standard should be replaced by a 25 μg/mL standard at high end of standard series accordingly. **Recommended dilution factors are listed. Some adjustment may need to be made empirically. 3. Note that the "reference cell" and "blank" are identical. A reference cell test sample is only required when using a double-beam UV-visible spectrophotometer for absorbance measurements. The reference position is that farthest from the front of the instrument, while the sample is placed in the position nearest to the front of the instrument. 4. Note that a dilution of the actual protein sample may be required for the resulting absorbance to fall within the linear range of the assay. Ask your TA or instructor for guidance on when to dilute your sample. 5. Allow each sample to incubate at room temperature for 10-30 minutes. (Record the actual incubation time in your notebook.) Be sure to cover each sample with parafilm and mix with inversion before absorbance measurements are made. 6. Measure the absorbance of each sample at 595 nm using a UV-visible spectrophotometer. Be sure to allow the instrument to warm up for at least 15 minutes prior to use. 7. Plot the absorbance of each BSA standard as a function of its theoretical concentration. The plot should be linear. 8. Determine the best fit of the data to a straight line in the form of the equation "y = mx + b" where y = absorbance at 595 nm and x = protein concentration. 9. Use this equation to calculate the concentration of the protein sample based on the measured absorbance. Note: If the absorbance of the test sample is outside of the absorbance range for the standards, then the assay must be repeated with a more appropriate dilution, if any. The linear range for the assay (and for most spectrophotometers is 0.2 - 0.8 O.D. units. 10. Select those samples containing substantial amounts of protein for SDS-PAGE. Content Vol. of Protein Sample Vol. of dI Water Volume of Bradford Protein Assay Reagent Dilution Factor ** Reference None 800 μL 200 μL Blank None 800 μL 200 μL 2.5 μg/mL BSA* 5 μL 795 μL 200 μL 5 μg/mL BSA 10 μL 790 μL 200 μL 10 μg/mL BSA 20 μL 780 μL 200 μL 15 μg/mL BSA 30 μL 770 μL 200 μL 20 μg/mL BSA 40 μL 760 μL 200 μL Lysate 50 μL 750 μL 200 μL 20-fold Lysate FT 50 μL 750 μL 200 μL 10-fold Binding FT 50 μL 750 μL 200 μL 2-fold Wash FT 50 μL 750 μL 200 μL None Elution Fraction #1 50 μL 750 μL 200 μL None Elution Fraction #2 50 μL 750 μL 200 μL None Elution Fraction #3 50 μL 750 μL 200 μL None3 SDS-PAGE 1. Clean and assemble the SDS-PAGE gel-casting frame. 2. Since the MW of T7 RNA polymerase is high, a lower concentration gel will be required here. So, pour an 8% resolving gel and a 5% stacking gel according to Table 2. Table 2. Preparation of Samples for SDS-PAGE Reagent 7% Resolving Gel Total = 5 mL 5% Stacking Gel Total = 2 mL Deionized Water Buffer 1.25 mL of 1.5 M Tris-HCl, pH 8.8 0.5 mL of 0.5 M Tris-HCl, pH 6.8 10% SDS 50 μL 20 μL 40% Acrylamide:Bisacrylamide Caution: Neurotoxin – do not ingest To be determined by student To be determined by student 10 % Ammonium Persulfate 50 μL 20 μL TEMED 20 μL 10 μL Adjust volumes of catalysts as needed. 3. Add 50 μL of 2-mercaptoethanol to 950 μL of Laemmli Sample Buffer for 1 mL of working sample buffer. Dilute 3 μL of the High MW marker with 6 μL of sample buffer to make a total of 9 μL; be prepared to only load 5-8 μL of this marker onto the gel. 4. Dilute 10 μL of each sample with 20 μL of sample buffer for a total of 30 μL each. Be prepared to only load ~20 μL of this sample onto the gel. 5. Boil the MW marker and protein samples for the gel for 2-3 min to fully denature the samples in the presence of SDS from the sample buffer. 6. Rinse out the wells of the gel and load the gel with each sample. Organize your samples according to the progression of the purification so that the resulting image will clearly indicate how well the purification proceeded from step to step. 7. Electrophorese the samples at 200 V until the dye front reaches the bottom of the gel, but has not run off of the gel. CAUTION: Use safety when performing electrophoresis; risk of electrocution exists. Turn off voltage before removing lid to electrohphoresis chamber to avoid mixing electricity and water. 8.