Biochemistry Laboratory Manual for CHEM 3131, Lecture notes of Biochemistry

Download Biochemistry Laboratory Manual for CHEM 3131 and more Lecture notes Biochemistry in PDF only on Docsity! 1 Second Edition Biochemistry Laboratory Manual for CHEM 3131 The University of Texas at El Paso Department of Chemistry Ricardo A. Bernal 2 Table of Contents Syllabus ..................................................................................................................................... 3 Lab Safety Rules ........................................................................................................................ 5 Commonly used Biochemical Standards .................................................................................... 6 Laboratory #1, Basic Pipetting and Buffer Dilutions .................................................................... 7 Lab Assignment ...................................................................................................................... 9 Laboratory #2, Polymerase Chain Reaction ..............................................................................10 New England Biolabs Protocol ..............................................................................................10 Protocol for Class ..................................................................................................................13 Laboratory #3, PCR Product Purification and Restriction Enzyme Digest. .................................15 Monarch® PCR & DNA Cleanup Kit (5 μg) Protocol (NEB #T1030).......................................15 Laboratory #4, Ligation and Bacterial Transformation ...............................................................18 Ligation Reaction ...................................................................................................................19 Transformation Protocol for BL21(DE3) Competent Cells ......................................................20 Transformation Protocol Variables .....................................................................................20 Laboratory #5, Protein Expression and Cell Lysis .....................................................................21 Cell Lysis ...............................................................................................................................22 Laboratory #6, Protein Purification using Column Chromatography ..........................................23 Column Chromatography.......................................................................................................25 While you wait for the Chromatography run to finish, please work on the following questions. ..............................................................................................................................................26 Laboratory #7, SDS-PAGE for Analytical Protein Gels ..............................................................28 Setting up the apparatus .......................................................................................................29 Removing and Staining the gel ..............................................................................................30 COOMASSIE BLUE STAINING .............................................................................................31 Laboratory #8, Protein Quantitation Methods (BCA Assay) .......................................................32 Pasco Wireless Spectrophotometer Instructions for Use .......................................................34 Laboratory #9, β-Galactosidase Activity Assay .........................................................................36 Laboratory #10, β-Galactosidase Enzyme Kinetics ...................................................................39 Laboratory #11, Kinetics Calculations, continuation from last week. ..........................................42 Appendix ...................................................................................................................................44 List of supplies for TAs ..........................................................................................................44 5 Lab Safety Rules 1. Always wear eye protection in the lab. 2. A lab coat MUST be worn in the lab always. 3. Don't Eat or Drink in Lab 4. Only authorized personnel are to be allowed into the lab (don’t bring your friends). 5. DO NOT BE WASTEFUL with reagents and supplies. 6. Dress appropriately (Closed toe shoes and long trousers must be worn in the lab. Sandals and shorts are not allowed.) 7. Clean up after yourself. Wash all your glassware and clean (disinfect) your work area. 8. Identify the Safety Equipment in case you need it in the future. 9. Don't Casually Dispose of Chemicals Down the Drain. There are special containers for everything. 10. Long hair must be tied back when using open flames. 11. Always wash your hands before leaving lab. 12. Excess reagents are never to be returned to stock bottles. 13. Always pour strong acids/bases into water and not the other way around. If you pour water into acid, the heat of reaction will cause the water to explode into steam, sometimes violently, and the acid will splatter. 14. If chemicals come into contact with your skin or eyes, flush immediately with copious amounts of water and consult and report the incident your TA. 15. Do not place backpacks or other personal items on the lab benches. 16. Keep the lab CLEAN (it should be spotless). 17. Treat all equipment with care!! 18. Before using an instrument, make sure you are trained properly. 19. Keep an eye out for instruments that act up. Notify someone immediately. 20. Be as careful for the safety of others as for yourself. Think before you act. Emergency Number 911(City of El Paso Police, Fire, EMS) UTEP Emergency Number 5611 (Campus Police) Poison Control Center 1-800-POISON-1(1-800-764-7661) Emergency Spill Response Coordinator EH&S Main Phone No. 747-7124 Facilities Services 7116 6 Commonly used Biochemical Standards 1.0 A260 unit ds DNA = 50 μg/mL = 0.15 mM (in nucleotides) The average molecular weight of an amino acid is 110 Da 1 mL water = 1 g/cm3 Standard Conditions • T = 25°C = 298K • P = 1 atm • Water 55.6 M • H+ conc. = 10-7 M (pH=7.0) How to make a solution from a concentrated Stock Solution • M1V1 = M2V2 where M=concentration and V=volume (make sure units match) Common Units • molarity (M, mM, μM, nM, pM, etc.) • normality (N, whose use is discouraged by IUPAC) • weight/volume (g/L, mg/mL, μg/mL, μg/μL, etc.) • percent weight or volume (%) • dilution factor (1000x, 10x, 1x, etc.) Common Terms and Notations • ATP adenosine 5’- triphosphate • DNA deoxyribonucleic acid • mM millimolar, 10-3 Molar, unit of concentration • ADP adenosine 5’- diphosphate • kDa kilodalton • μM micromolar, 10-6 Molar, unit of concentration • E. coli Escherichia coli • Apo nucleotide-free, unbound state • in vivo within the living • EM electron microscopy • PCR polymerase chain reaction • EDTA ethylene-diamine-tetraacetic acid • HPLC high-performance liquid chromatography • pI isoelectric point • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis • OD600 optical density, 600 nanometer wavelength • DTT dithiothreitol, Cleland’s reagent • mg/ml milligrams/milliliter, 10-3g/10-3L, unit of concentration • PDB protein databank • in vitro within the test tube • 2xTY yeast, tryptone and salt-based growth medium • IPTG isopropyl β-D-1-thiogalactoside • μM micromolar, 10-6 Molar, unit of concentration • HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid • DNase deoxyribonucleic acid hydrolase • SEC size-exclusion chromatography • μg microgram, 10-6 gram, unit of weight • BCA bicinchoninic acid 7 Laboratory #1, Basic Pipetting and Buffer Dilutions Objectives 1. To learn how to properly measure solution volumes using serological pipets and micro-pipettors. 2. Learn to measure solutions of varying viscosity accurately. 3. Learn to prepare the following solutions from stock solutions. a. 50 mL of TE Buffer (for DNA) containing i. 10 mM Tris-HCL pH 8.0 ii. 1 mM EDTA pH 8.0 b. 50 mL Borate buffer for DNA gels Experimental procedure 1) You will work in groups of three so select your partners. Do not start on anything until given instructions by the TA. 2) First, you need to go to your TA to check out the needed supplies. a. Serological Pipets Pipettes (one 5 mL and one 10 mL) b. One Serological Pipet bulb c. One set of Micropipettors (P20, P200 & P1000) d. One box each of P1000 and P200 tips e. Two 50 mL conical tubes (with purple cap) f. One microcentrifuge tube 3) Your TA will give you instructions on the operation of the Micropipettors and serological pipet bulbs. Please make notes on their proper use and ask plenty of questions. 4) You will now figure out how to pipet the following volumes for a dry run. Ask TA if unsure a. 539 μl b. 233 μl c. 143 μl d. 27 μl 5) Weigh your microcentrifuge tube and write down the value on a piece of paper (2 significant figures) or on the tube itself with a sharpie. 6) You will now pipet the following into the pre-weighed microcentrifuge tube to create a mixture (you will mix solutions A through D in the microcentrifuge tube). a. 539 μl of solution labeled A (water) b. 233 μl of solution labeled B (glycerol) c. 143 μl of solution labeled C (ethanol) d. 27 μl of solution labeled D (acetonitrile) 10 Laboratory #2, Polymerase Chain Reaction Objectives 1. To learn how to perform Polymerase Chain Reaction (PCR) for the amplification of DNA for future recombinant protein production. 2. To analyze the results of PCR using DNA gel electrophoresis. Introduction This semester, you will be cloning β-galactosidase. Part of the cloning procedure is to first amplify the gene of interest so that you can have a sufficient quantity to manipulate it further (purification, digestion of restriction sites, etc.). PCR is very important and a key component of modern molecular biology. For your lab report, you need to find the DNA sequence for β- galactosidase online. You also should design the forward and reverse primers (include Nde I and Xho I restriction sites) to be used in the PCR reaction. For today’s experiment, you will be given the PCR primers and the DNA template but you need to find those sequences yourself for the report. Experimental procedure (Protocol begins on page 13) • The above named β-galactosidase primers were designed and purchased from IDT. • TA made 100 µL of a 10 µM stock of each for you as indicated below. Primer # Concentration of Stock DNA Volume of DNA Volume of water Final concentration of primer 1 100 µM 10 µL 90 µL 10 µM 2 100 µM 10 µL 90 µL 10 µM New England Biolabs Protocol The following protocol was taken from New England BioLabs website https://www.neb.com/protocols/2012/08/29/protocol-for-q5-high-fidelity-2x-master-mix-m0492 Please note that protocols with Q5 High-Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance. Reaction Setup: We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to use. See table below 11 Component 25 µl Reaction 50 µl Reaction Final Concentration Q5 High-Fidelity 2X Master Mix 12.5 µl 25 µl 1X 10 µM Forward Primer 1.25 µl 2.5 µl 0.5 µM 10 µM Reverse Primer 1.25 µl 2.5 µl 0.5 µM Template DNA variable variable < 1,000 ng Nuclease-Free Water to 25 µl to 50 µl Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Transfer PCR tubes to a PCR machine and begin thermocycling. Thermocycling Conditions for a Routine PCR: STEP TEMP TIME Initial Denaturation 98°C 30 seconds 25–35 Cycles 98°C 5–10 seconds *50–72°C 10–30 seconds 72°C 20–30 seconds/kb Final Extension 72°C 2 minutes Hold 4–10°C *Use of the NEB Tm Calculator is highly recommended. General Guidelines: Template: Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 µl reaction are as follows: DNA AMOUNT DNA Genomic 1 ng–1 µg Plasmid or Viral 1 pg–1 ng 1. Primers: Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 can be used to design or analyze primers. The best results are typically seen when using each primer at a final concentration of 0.5 µM in the reaction. 12 2. Mg++ and additives: The Q5 High-Fidelity Master Mix contains 2.0 mM Mg++ when used at a 1X concentration. This is optimal for most PCR products generated with this master mix. 3. Deoxynucleotides: The final concentration of dNTPs is 200 μM of each deoxynucleotide in the 1X Q5 High-Fidelity Master Mix. Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and is not recommended for use with uracil-containing primers or templates. 4. Q5 High-Fidelity DNA Polymerase concentration: The concentration of Q5 High-Fidelity DNA Polymerase in the Q5 High-Fidelity 2X Master Mix has been optimized for best results under a wide range of conditions. 5. Denaturation: An initial denaturation of 30 seconds at 98°C is sufficient for most amplicons from pure DNA templates. Longer denaturation times can be used (up to 3 minutes) for templates that require it. During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5–10 second denaturation at 98°C is recommended for most templates. 6. Annealing: Optimal annealing temperatures for Q5 High-Fidelity DNA Polymerase tend to be higher than for other PCR polymerases. The NEB Tm Calculator should be used to determine the annealing temperature when using this enzyme. Typically use a 10–30 second annealing step at 3°C above the Tm of the lower Tm primer. A temperature gradient can also be used to optimize the annealing temperature for each primer pair. For high Tm primer pairs, two-step cycling without a separate annealing step can be used (see note 10). 7. Extension: The recommended extension temperature is 72°C. Extension times are generally 20–30 seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for simple templates (plasmid, E. coli, etc.) or complex templates < 1 kb. Extension time can be increased to 40 seconds per kb for cDNA or long, complex templates, if necessary. A final extension of 2 minutes at 72°C is recommended. 8. Cycle number: Generally, 25–35 cycles yield sufficient product. For genomic amplicons, 30-35 cycles are recommended. 9. 2-step PCR: When primers with annealing temperatures ≥ 72°C are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible. 10. Amplification of long products: When amplifying products > 6 kb, it is often helpful to increase the extension time to 40–50 seconds/kb. 11. PCR product: The PCR products generated using Q5 High-Fidelity 2X Master Mix have blunt ends. If cloning is the next step, then blunt-end cloning is recommended. If T/A-cloning is preferred, the DNA should be purified prior to A-addition, as Q5 High-Fidelity DNA Polymerase will degrade any overhangs generated. 15 Laboratory #3, PCR Product Purification and Restriction Enzyme Digest. Objectives 1. To learn how to purify DNA. 2. To learn how to use restriction enzymes to create “sticky ends” for subsequent ligation reactions where the gene of interest is inserted into a plasmid. 3. To analyze the results of Restriction Enzyme digests using DNA gel electrophoresis. Introduction Nucleic acids have the property of being able to selectively bind to silica. An excellent source of information about the process of purifying DNA using silica is the following website. http://bitesizebio.com/13516/how-dna-extraction-rna-miniprep-kits-work . After purification of your nucleic acid, your purified DNA (either plasmid or PCR product) is ready for restriction enzyme digestion. Restriction enzymes are used because they leave overhangs in one of the strands which can be used to ligate to another strand that had also been digested with the same restriction enzyme. This overhang adds specificity to the ligation so that the gene of interest is inserted into a plasmid in a specific direction only. Experimental procedure Spin Column Purification of PCR product From New England Biolabs (Recommended protocol) https://www.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol Monarch® PCR & DNA Cleanup Kit (5 μg) Protocol (NEB #T1030) General Guidelines: Input amount of DNA to be purified should not exceed the binding capacity of the column (5 μg). A starting sample volume of 20–100 μl is recommended. For smaller samples, TE can be used to adjust the volume to the recommended volume range. Centrifugation should be carried out at 16,000 x g in a standard laboratory microcentrifuge at room temperature. Buffer Preparation: Always keep all buffer bottles tightly closed when not in use. All centrifugation steps should be carried out at 16,000 x g. (~13K RPM in a typical microcentrifuge). This ensures all traces of buffer are eluted at each step. 1. Dilute sample with DNA Cleanup Binding Buffer according to the table below. Mix well by pipetting up and down or flicking the tube. Do not vortex. A starting sample volume of 20–100 μl is recommended. For smaller samples, TE can be used to adjust the volume. For diluted samples larger than 800 μl, load a portion of the sample, proceed with Step 2, and then repeat as necessary. 16 SAMPLE TYPE RATIO OF BINDING BUFFER: SAMPLE EXAMPLE dsDNA > 2 kb (plasmids, gDNA) 2:1 200 μl:100 μl dsDNA < 2 kb (some amplicons, fragments) 5:1 use this ratio!! 500 μl:100 μl ssDNA (cDNA, M13) 7:1 700 μl:100 μl 2. You will do the 5:1 ratio for your PCR and Vector RE Digestions 3. Insert column into collection tube and load sample onto column and close the cap. Spin for 1 minute, then discard flow-through. 4. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional. 5. Repeat wash (Step 3). 6. Transfer column to a clean 1.5 mL microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to next step. 7. Add 35 μl of 1 mM Tris Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA. Note: Typical elution volumes are 6–20 μl. Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA (≥ 10 kb), heating the elution buffer to 50°C prior to use can improve yield. Care should be used to ensure the elution buffer is delivered onto the matrix and not the wall of the column to maximize elution efficiency. Normally, you would determine the concentration of the purified DNA spectrophotometrically by checking the absorbance of the DNA at a wavelength of 280nm. However, to save time you can just digest your entire 35 μl in the restriction enzyme digest in the next step. Restriction Enzyme Digest of PCR Product to create overhang ends 1. Restriction Enzyme Digest of PCR Reaction Product Amount Component ______ µL DNA _______ µg/µl purified PCR product 5 µL 10X Buffer ________ µL Water 1 µL Enzyme #1 Xho I of 1:10 dilution in 1x Buffer 1 µL Enzyme #2 Nde I of 1:10 dilution in 1x Buffer 50 µL Total Volume 17 2. pET-30a Plasmid Vector Amount Component 7 µL DNA 1.0 µg/µl pET30a vector 5 µL 10X Buffer ________ µL Water 1 µL Enzyme #1 Xho I of 1:10 dilution in 1x Buffer 1 µL Enzyme #2 Nde I of 1:10 dilution in 1x Buffer 50 µL Total Volume • Keep your DNA on ice. • Make sure your tubes are labeled and give it to your TA • Your TA will do the following for you so that it is ready for next week • Incubate the RE reactions at 37 degrees for ~3 hours. • Heat inactivate the reactions to denature the restriction enzymes (~70⁰C for about 15 minutes) • Please return all supplies and equipment to your TA. • Finally, you should wipe down your bench with ethanol provided 20 5. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells. Transformation Protocol for BL21(DE3) Competent Cells Adapted from https://www.neb.com/protocols/1/01/01/transformation-protocol-for-bl21-de3- competent-cells-c2527 1. Thaw a tube of BL21(DE3) Competent E. coli cells on ice for 10 minutes. It is critical that the tube is never removed from the ice or warmed up! 2. Add 1–5 µl containing 1 pg–100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4–5 times to mix cells and DNA. Do not vortex. 3. Keep the mixture on ice for 30 minutes. Do not mix. 4. Heat shock at exactly 42°C for exactly 10 seconds. Do not mix. 5. IMMEDIATELY place on ice for exactly 5 minutes. Do not mix. 6. Pipette 950 µl of room temperature SOC media into the mixture. 7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate. 8. Warm two LB + 100 µg/mL Ampicillin plates to 37°C. 9. Mix the cells thoroughly by flicking the tube and inverting. 10. Spread 50 & 100 µl of cells onto each selection plate with a sterile spreader and incubate overnight at 37°C. Transformation Protocol Variables Thawing: Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0°C will decrease the transformation efficiency. Incubation of DNA with Cells on Ice: For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes you shorten this step. Heat Shock: Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 10 seconds at 42°C is optimal. Outgrowth: Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes you shorten this step. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking. Plating: Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation. • Please return all supplies and equipment to your TA. • Finally, you should wipe down your bench with ethanol provided 21 Laboratory #5, Protein Expression and Cell Lysis Objectives 1. To learn how to express recombinant proteins in bacteria. 2. To learn how to lyse bacteria to extract your protein of interest. Introduction In molecular biology (genetic engineering), it is critical to be able to induce a cell (typically bacteria) to express or produce a protein from a gene that was introduced into that cell via a vector (carrier). In our case, it is a plasmid carrying the gene for β-galactosidase. That plasmid also contains a selectable marker typically in the form of antibiotic resistance. Any cell that gets a plasmid will become resistant to that antibiotic and all cells that did not get the plasmid will be killed off. The plasmid also contains the gene of interest under the control of an inducible promotor. In our case, the promotor is part of the lactose operon and is responsible for the induction of β-galactosidase when the cell detects the presence of lactose. In other words, lactose induces the production of β-galactosidase (an enzyme that degrades lactose). Instead of adding lactose to the bacterial culture to induce production of our protein, we add a non- hydrolyzable lactose analog so that its concentration remains constant. After protein production, the cells are harvested by centrifugation and then lysed. Once lysed, the cellular debris is removed before the protein is purified from the crude extract. Cell lysis is accomplished using lysozyme, an enzyme that digests the peptidoglycan layer of the cell well. The cells are then placed in a hypotonic solution so that the cells burst. Experimental procedure To save time, your TA selected a colony from your plates and inoculated a liquid culture in 250 mL of media called 2XTY (see recipe below). This must be done early in the morning because it takes the cells about 8 hours to grow to log phage and to an Optical Density (OD) of 0.6 at 600 nm. You will begin todays experiment at Cell Lysis step below. Prepare cells and Induce protein production: 1. Prepare a 50 mL of 2XTY containing 100 µg/mL ampicillin in a baffled flask for increased aeration. 2XTY For 1 Liter Tryptone 16 g Yeast Extract 10 g NaCl 5 g Add 2 drops of 10 M NaOH to adjust the pH to 7.0 Autoclave for 20 minutes at 121.1⁰C 2. Pick a single isolated colony from last week’s plate and inoculate the media. 3. Incubate the culture at 37 ºC with shaking at 220 rpm for ~ 8 hours until the optical density at 600 nm (OD600) is between 0.6-0.8. 22 4. Induce protein production after the OD600 has reached the indicated OD by adding 1 M IPTG to a final concentration of 1mM. This is just a simple 1:1000 dilution. 5. Protein induction is to proceed for 2 hours at a reduced temperature of 25 ºC. Keep flask shaking 220 rpm for proper aeration. Cell Lysis 1. After induction, spin down cells at 6000x g for 30 minutes at 4 ºC. 2. Remove the supernatant (old spent media) with a Pipetman and discard. 3. Resuspend the pellet/cells in a final volume of 1 mL Lysis Buffer for each 50 mL original culture. Lysis Buffer For 1 L 10 mM HEPES pH 7.5 10 mL of 1 M 50 mM EDTA 100 mL of 0.5 M 0.02 % Sodium Azide 1 mL of 20% Stock 4. Add 10 μl of Lysozyme solution to the resuspended cells and gently mix end-over-end every minute for 30 minutes at 4 ºC. 5. Freeze at -20 ⁰C until frozen. 6. Thaw the tube by gently mixing end-over-end every minute at room temperature until thawed. 7. Repeat step 4-5 for a total of 3 cycles of freeze/thaw. 8. Once sample has become thick and extremely viscous then the cells are lysed. Otherwise you need to repeat freeze/thaw until sample has become viscous. 9. Add 10 μl of DNase I solution and 60 mM MgCl2. 10. Gently mix end-over-end every minute for 30 minutes at 4 ºC. Sample should go from viscous to liquid again as DNA is degraded. 11. Transfer sample to a high speed centrifuge tube spin down at maximum speed for 30 minutes. 12. Carefully decant the supernatant into a new microcentrifuge tube. Discard cellular debris pellet. 13. Freeze supernatant containing protein of interest at -80 ºC for purification next week. 14. Please return all supplies and equipment to your TA. 15. Finally, you should wipe down your bench with the ethanol provided. 25 Most modern column chromatography is now computer controlled and proteins are monitored using UV absorption as they elute from the column. In today’s lab, we will use an AKTA Start chromatography system. The results of the protein purification will be analyzed next week. Experimental procedure Column Chromatography 1. Your TA has thawed out your crude extract from the -80 ºC freezer from last week and has combined the samples for loading on to the chromatography column this week. 2. Before loading the sample on the column, we must spin down insoluble debris on micro- centrifuge at max speed for 5 minutes. 3. Dilute the sample 20-fold in Chromatography Buffer A. 4. Load the total volume onto a High Trap Q HP 5 ml Column (a strong anion exchange column). 5. The chromatography run is completely automated (apart from sample loading) and has been programmed to wash the column with a large volume of Chromatography Buffer A so that any unbound proteins are completely removed. 6. The computer will monitor the UV280nm and the conductivity of the buffer coming out of the column. This data is recorded and graphed on the screen for you. 7. β-galactosidase should elute at a conductivity of about 7.0- 8.0 mS/cm and so you should look for a corresponding UV280nm peak at about this same area. 8. Save the fractions by capping the tubes of interest and make sure to label the tubes. 9. Please return all supplies and equipment to your TA. 10. Finally, you should wipe down your bench with the ethanol provided. Chromatography Buffer A For 500 mL Chromatography Buffer B For 500 mL 10 mM HEPES 5 ml of 1 M 10 mM HEPES Buffer 5 ml of 1 M 5 mM EDTA 5 ml of 0.5 M 5 mM EDTA 5 ml of 0.5 M No NaCl 0 ml of 5 M 1 M NaCl 100 ml of 5 M 26 While you wait for the Chromatography run to finish, please work on the following questions. 1. Predict the elution sequence of the listed compounds on anion exchange, cation exchange and hydrophobic interaction chromatography at pH 7.0. Arg Cys-Lys-Arg-Gly Glu-Lys Leu-Phe-Ala Phosphorylated Tyr Trp-Val-Phe Hydrophobic Interaction Column 1. ________________ 2. ________________ 3. ________________ 4. ________________ 5. ________________ 6. ________________ Anion Exchange Column 1. ________________ 2. ________________ 3. ________________ 4. ________________ 5. ________________ 6. ________________ Cation Exchange Column 1. ________________ 2. ________________ 3. ________________ 4. ________________ 5. ________________ 6. ________________ 27 2. What type of column would you choose if your protein has the following characteristics? Characteristic Column Type to Use The protein is rich in glutamate and has a molecular weight of 30 kDa The protein is rich in tryptophan and has a molecular weight of 20 kDa The protein is rich in lysine and has a molecular weight of 50 kDa The protein has a molecular weight of 150 kDa and is isoelectric at pH 7 The protein is rich in cysteine and has a molecular weight of 30 kDa 3. You find that your protein sample loses activity during storage but looks fine on an SDS-PAGE. What can you do about this? Circle correct answer a) Add many more additional purification steps b) Use a protease inhibitor during purification steps c) Perform each step as quickly as possible, in a cold-room d) Be careful not to damage your protein during purification e) All of the above 4. Your protein is large and has all amino acids represented in its sequence. The isoelectric point is exactly at the pH which you normally use to purify proteins (pH 7.5). Figure out a strategy to be able to use ion exchange chromatography with this protein. 30 e. Set the electrode assembly to the open position on a clean, flat surface (A). f. Place the gel cassettes into the electrode assembly. Use the buffer dam (included with the cell) to complete the assembly. i. Place the first cassette with the short plate facing inward and so the gel rests at a 30° angle away from the center of the electrode assembly. Make sure the electrode assembly remains balanced and does not tip over. ii. Place the second gel or buffer dam on the other side of the electrode assembly, again by resting the gel on the supports. The gels rest at 30° angles, one on either side of the electrode assembly, tilting away from the center of the frame (B). g. Gently push both gels toward each other, making sure that they rest firmly and squarely against the green gasket that is built into the electrode assembly. Align the short plates to ensure the edge sits just below the notch at the top of the green gasket (C). h. While gently squeezing the gel cassettes (or cassette and buffer dam) against the green gaskets (maintaining constant pressure and with both gels in place), slide the green arms of the clamping frame one at a time over the gels, locking them into place (D,E). i. The wing clamps of the electrode assembly lift each gel cassette up against the notch in the green gasket, forming a seal. Check again that the short plates sit just below the notch at the top of the green gasket (C). j. Place the electrophoresis module into the tank (F) and fill the buffer chambers with 1x running buffer: i. Fill the inner buffer chamber completely to the top ii. Add most if not all the remaining buffer to the outer buffer chamber 5. Load samples and run the gels at a constant voltage of 200V. 6. Stop the run when the dye front reaches 0.5 cm from the bottom of the gel. Removing and Staining the gel 1. After electrophoresis is complete, turn off the power supply and disconnect the electrical leads. 2. Remove the lid from the tank and remove the gels from the cell. Pour off and discard the running buffer. 31 3. To open the cassette, align the arrow on the opening lever with the arrows marked on the cassette and insert the lever between the cassette plates at indicated locations. Apply downward pressure to break each seal. Do not twist the lever. 4. Pull the two plates apart from the top of the cassette, and gently remove the gel by teasing it with the pipet tip and drop it into the staining solution. a. Do not touch the gel with your hands because you will contaminate it! b. Stain the gel for a minimum of 1 hour. c. De-stain overnight or until desired intensity is achieved. 5. Please return all supplies and equipment to your TA. 6. Finally, you should wipe down your bench with the ethanol provided. COOMASSIE BLUE STAINING Detection of protein bands in a gel by Coomassie blue staining depends on nonspecific binding of a dye, Coomassie brilliant blue R, to proteins. The detection limit is 0.3 to 1 µg/protein band. In this procedure, proteins separated in a polyacrylamide gel are precipitated using a fixing solution containing methanol/acetic acid. The location of the precipitated proteins is then detected using Coomassie blue (which turns the entire gel blue). After de-staining, the blue protein bands appear against a clear background. The gel can then be stored in acetic acid or water, photographed, or dried to maintain a permanent record. Coomassie blue staining solution 50% (v/v) methanol 0.05% (w/v) Coomassie brilliant blue R-250 10% (v/v) acetic acid 40% H2O Fixing solution for Coomassie blue 50% (v/v) ethanol 10% (v/v) acetic acid 40% H2O Store at room temperature Methanol/acetic acid de-staining solution 40% (v/v) ethanol 5% (v/v) acetic acid Store at room temperature 32 Laboratory #8, Protein Quantitation Methods (BCA Assay) Objectives 1. To learn how to determine the concentration of an unknown protein. 2. To perfect pipetting skills 3. To analyze the results of the concentration assay using an EXCEL spreadsheet. Introduction The bicinchoninic acid (BCA) assay was developed by Paul Smith at the Pierce Chemical Company in 1985 and is used to determine the total protein concentration in a solution. Protein is determined by a color change from light green to purple depending on the amount of protein present. There are two reactions that occur in the assay that result in the color change. First, the peptide bonds of a protein will reduce Cu2+ ions to Cu+ ions at an elevated temperature (between 40-60 ⁰C). The amount of Cu2+ ions reduced is directly proportional to the amount of protein present. In the Second reaction, bicinchoninic acid will chelate the resulting Cu+ ions which produces the purple colored complex BCA-Cu+. The purple color that is developed can be quantitated using absorption of light at 562 nm in a spectrophotometer. The unknown concentration of a protein must be compared to a known concentration of a control protein and so a standard curve must first be generated. The absorption of the unknown protein is then compared to the standard curve to estimate the concentration of the unknown. The control protein that is usually chosen is bovine serum albumin because it is a very stable and well known protein. Experimental procedure 1. You will be working as usual in groups of three and you will need the following items from your TA. a. BSA standard b. Reagent A and B for the BCA assay c. Cuvettes for the spectrophotometer d. A portable PASCO wireless spectrophotometer i. Download the software from the following website (for phone laptop or tablet) ii. https://www.pasco.com/downloads/spectrometry/index.cfm e. Pipettors and tips f. 15 microcentrifuge tubes and a tube rack g. A bottle of deionized water h. One 50 ml conical tube. i. Two spectrophotometer cuvettes (one for standards and one for unknowns) 2. Label your microcentrifuge tubes A-K and 1-4 as indicated in the Standards and Unknowns table below. 3. Prepare a set of protein standards of known concentration by adding everything as listed in the “Standards” table below and in the order listed here. a. First pipet out the diluent (diH2O) into all the tubes. b. Second, pipet the BSA standard (or unknown protein) into appropriate tubes. c. Finally, prepare and pipette the working reagent. 35 green light icons and clear sides face the white light and absorbance spectrum icons. e. Select CALIBRATE REFERENCE from the menu at the bottom of the screen. A check mark will appear when the calibration is complete. 3. Finding the 562 nm Wavelength to Analyze a. Place the cuvette into the spectrometer as you did in the calibration. b. Select the red RECORD circle at the bottom left of the screen to start analyzing the solution. (It changes into a square while data is being collected.) c. Select the red STOP RECORDING square to stop data collection. d. Select SCALE TO FIT to rescale your data. e. Use the ADD COORDINATE to locate the 562 nm wavelength to analyze on the curve. f. A small hand will replace your cursor. Move it to the box that has appeared on the graph and drag the box slowly toward the curve. As you get near the curve an arrow will appear that indicates a specific wavelength on the curve. Releasing the box will snap the box to the point on the curve the arrow is pointing to. g. Drag the box along the curve to find the 562 nm wavelength to analyze. 4. Record the absorbance value in the table above and repeat the procedure for all unknown and known solutions you prepared. 5. Please return all supplies and equipment to your TA. 6. Finally, you should wipe down your bench with the ethanol provided. 36 Laboratory #9, β-Galactosidase Activity Assay Objectives 1. To learn how to check that a protein you purified is biologically active. 2. To learn about the proper conditions that are required to get biological activity. 3. To analyze and interpret the results of the activity assay Introduction Enzymes are the biological version of catalysts in that they accelerate the rate of a biological reaction by reducing the activation energy barrier that is required for the reaction to proceed. Just like small molecule catalysts, enzymes are not consumed in the reaction and are available to be reused repeatedly in many reactions. Enzymes like most other proteins are globular in shape and can also be denatured which will render them inactive. To ensure that you are working with a native and fully active protein, you can check for biological activity after the enzyme has been purified. To check for activity, you must know certain characteristics about the enzyme to create a suitable activity assay. First you must know what the natural substrate is for this enzyme. You can utilize either the natural substrate or a derivative the closely mimics the natural substrate but gives you an easy way to detect the product whereas the natural substrate cannot be detected. For example, in today’s lab, we will be using ortho-Nitrophenyl-β- galactoside (ONPG) instead of the natural substrate for β-Galactosidase in E. coli, lactose. Cleavage of the disaccharide lactose results in one D-galactose and one D-glucose. Both cleavage products are colorless and difficult to detect as lactose cleavage products. However, ONPG (is colorless) cleavage produces D-galactose and O-nitrophenyl. The O-nitrophenyl as a bright yellow color that can easily be detected by a spectrophotometer at a wavelength of 420nm. If you can detect the appearance of yellow ONP and the absence of yellow color in the negative control, then you can be confident that your enzyme, in this case β-Galactosidase, is active. Keep in mind that different enzyme purifications will have different levels of activity, indicating varying levels of native/denatured enzyme. Some preparations will be bad while others will be excellent. This assay will only tell you if there is active enzyme present in your purified protein and not the quality of the preparation. Next week, we will be doing kinetics which is much more complex but is also more qualitative. 37 Experimental procedure We will again be using the PASCO spectrophotometers for this week’s lab. Please refer to the set up and use in last week’s lab write-up (Section labeled “Pasco Wireless Spectrophotometer Instructions for Use”). 1. You will be working as usual in groups of three and you will need the following items from your TA. a. A portable PASCO wireless spectrophotometer b. You will need an aliquot of i. Purified β-Galactosidase enzyme (0.08 mg/ml) ii. ONPG (1 mg/ml (3.3mM)) supplied as a 10 X stock iii. Reaction buffer (10mM HEPES pH 7.5, 50 mM NaCl, 5 mM EDTA, 10mM MgCl2) c. Pipettors and tips d. 2 microcentrifuge tubes and a tube rack i. Label these - control, and experimental e. Two spectrophotometer cuvettes (one each for negative control and experimental). i. Label these - control and experimental. Remember to label these on the rough side and not the side with  symbol. 2. First, please connect your spectrophotometer to your computer. You will not have time to set this up after you mix your reaction components so do it first. a. It is going to be critical that you begin collecting data as soon as you mix your components because the reactions occur remarkably fast (E. coli β- Galactosidase can carry out as many as 400 reactions per second at room temperature). If you don’t start collecting data immediately, you will miss the reaction! b. You will be using the timed run for your spectrophotometer readings. Set it up to collected data for 10-15 minutes with 10 second intervals. c. In the software, go to Analyze solution. Collect the dark reference and a light reference (just use air). Then go to TIME and start recoding when ready. 3. You will do your negative control first (it will also serve as practice). a. Add 950 μl Reaction buffer to a labeled cuvette. b. Add 0 μl Purified β-Galactosidase enzyme and mix well. c. Place cuvette in spectrophotometer and begin reading as soon as you add the ONPG in step d below. d. Add 50 μl ONPG (10 mg/ml) supplied as a 10 X stock and mix well by pipetting up and down several times. e. Once the run has started, do NOT touch the spectrophotometer. Even bumping it will give you altered readings. f. Once the run is finished hit the stop record button and export the file to a csv file which you can open with excel. Please create a plot of your data for your report. 4. Next you will do your experimental reading. 40 b. You will be using the timed run for your spectrophotometer readings. Set it up to collected data for 10-15 minutes with 10 second intervals. c. In the software, go to Analyze solution. Collect the dark reference and a light reference (just use air). Then go to TIME and start recoding when ready. 3. To calculate initial velocities (Vo), each group will run 6 reactions as listed in the table below (if there are not enough groups then only do the first sets of reactions listed). a. Results from the entire class will be pooled together to calculate the kinetics values. Be sure to ask the TA about which reactions you are responsible for before you start so that you do not replicate date that someone else is also doing. i. Add the reaction buffer to the cuvette first ii. Next add the β-Galactosidase enzyme and mix well iii. The substrate will initiate the reaction and so you must do this as quickly as possible. 1. Place the cuvette in the spectrophotometer 2. Take the appropriate amount of ONPG and put it into a microcentrifuge tube (NOT the cuvette with the enzyme!). 3. Add the inhibitor to the ONPG and mix it well. 4. Now pipet the ONPG/inhibitor mixture with a P200 pipettor set to 200 μl and quickly mix it into the reaction mixture by pipetting up and down three times. DO NOT MAKE BUBBLES/FOAM 5. Collect data for about 10 minutes and do not touch the cuvette or spectrophotometer because it may influence your results. 6. After you stop the recording, export your data as a tube#.csv file (which can be read by excel). 7. Move on to the next tube and proceed as above. Experimental setup table is on the next page 41 Experimental Reaction Setup Tube # Reaction Buffer (μl) β−Galactosidase (μl) ONPG (μl) Inhibitor (IPTG) (μl) Resulting Vo 1 993 2 5 0 2 988 2 10 0 3 978 2 20 0 4 968 2 30 0 5 958 2 40 0 6 948 2 50 0 7 983 2 5 10 8 978 2 10 10 9 968 2 20 10 10 958 2 30 10 11 948 2 40 10 12 938 2 50 10 13 973 2 5 20 14 968 2 10 20 15 958 2 20 20 16 948 2 30 20 17 938 2 40 20 18 928 2 50 20 19 963 2 5 30 20 958 2 10 30 21 948 2 20 30 22 938 2 30 30 23 928 2 40 30 24 918 2 50 30 25 953 2 5 40 26 948 2 10 40 27 938 2 20 40 28 928 2 30 40 29 918 2 40 40 30 908 2 50 40 31 943 2 5 50 32 938 2 10 50 33 928 2 20 50 34 918 2 30 50 35 908 2 40 50 36 898 2 50 50 4. For data analysis, you need to plot your data in excel and then calculate a best fit line through the data (I would recommend that you use linear regression). The slope of that line is your initial velocity. We will need an initial velocity data point from each one of the experimental runs in the tables above to calculate the kinetics data. 5. The lab for next week will include a tutorial on the data analysis for Km and Vmax calculations. Therefore, you will not need to turn in a lab report until the end of the next lab. In other words, we are combining the lab report from this week with next week’s lab. a. If you want to get ahead for next week, you can download and try out the excel spreadsheet called “Lineweaver_Burk_Template.xlsx” from Blackboard. b. Make sure you bring a laptop with excel with you next week! 6. Please return all supplies and equipment to your TA. 7. Finally, you should wipe down your bench with the ethanol provided. 42 Laboratory #11, Kinetics Calculations, continuation from last week. Objectives 1. To learn how to calculate the initial velocity from the absorption data collected last week. 2. To learn how to calculate the Km and Vmax using excel 3. To analyze the results and determine the effect of the inhibitor IPTG. Introduction This week’s lab is a continuation of last week. We will use the data collected last week to calculate the kinetics values Km and Vmax. This is a relatively simple idea but it is data heavy which can lead to some confusion. Therefore, a spreadsheet has been created for you to help guide you along the data analysis pathway. You’ll be walked through the data analysis in class. Experimental procedure 1. There is no wet lab this week. You need a laptop computer (or take very good notes) that has excel installed on it so you can follow along as everything is explained to you. 2. Please download the spreadsheet named “Lineweaver_Burk_Template.xlsx” from Blackboard. 3. There are two spreadsheets in the excel file. The first one is for calculating the Vo and the second brings all the data from the entire class to plot a Lineweaver-Burk plot. DO NOT MAKE CHANGES TO ANYTHING OTHER THAN THE YELLOW CELLS! 4. First, use your data from last week to calculate the six Vo values for your group. a. Open the tube-1.csv file in excel for your first reaction from last week. b. Copy the absorbance values and paste them into the yellow column on the Vo calculation sheet. c. Use only the linear part at the beginning of the reaction. You will therefore have to adjust the formula to use fewer cells. Adjust the formula by putting the cursor on cell F23. Then modify the values underlined here [=INDEX(LINEST(C2:C14,A2:A14),1)]. Leave the rest of the formula alone! d. The slope of the line is equal to the initial velocity Vo and shows up automatically at F23. e. Enter the Vo value you get in the table below and then repeat the same procedure for your other reactions until you have calculated all the Vo values for your group. 5. Once all the Vo values have been determined, switch to the Lineweaver-Burk spreadsheet by clicking on the second tab at the bottom of the Excel program window. 6. Now fill in the yellow cells with the initial velocity Vo values you just calculated. Fill in all others with the appropriate Vo values from all the other groups in you class. 7. The Lineweaver-Burk plot is plotted automatically. The Km and Vmax values are also calculated automatically and show up in the cells L22 and L23. 8. You can also determine the type of IPTG inhibition by checking the Lineweaver-Burk plot and how the lines behave relative to each other. 45 Laboratory #3 Required Supplies and Equipment For TA: Supplies Needed per lab section of 18 students. 6 groups of 3 1. Spray bottle with Ethanol 2. UV Spectrophotometer (use the one in analytical lab) 3. 3 microcentrifuges (2 new Eppendorf and one old BioRad) 1. Xho I and Nde I Restriction Enzymes 2. 6 ice buckets with ice 4. Spin column kits for DNA purification (including spin columns and all associated buffers) 5. Waste containers for tips etc. (6) 6. 1.5 mL tubes racks (6) 7. 37⁰C heat block 8. Microcentrifuge tubes 9. 6 sets of micro pipettors (P1000, P200, P20). 7th set of micro pipettors is for TA 10. 6 sets of micropipettor tips (P1000 and P200) Preferably the ART tips to avoid pipet contamination. 11. P10 micro pipettors (2) and tips. Laboratory #4 Required Supplies and Equipment For TA: Supplies Needed per lab section of 18 students. 6 groups of 3 1. Spray bottle with Ethanol 2. Waste containers for tips etc. (6) 3. UV Spectrophotometer (use the one in analytical lab) 4. 3 microcentrifuges (2 new Eppendorf and one old BioRad) 5. 65⁰C heat block 6. 6 ice buckets with ice 7. 37⁰C incubator (already in that room) 8. 50 mL conical tube racks (6) 9. 1.5 mL tubes racks (6) 10. Bag of 1.5 mL Eppendorf tubes 11. 6 sets of micro pipettors (P1000, P200, P20). 7th set of micro pipettors is for TA 12. 6 sets of micropipettor tips (P1000 and P200) Preferably the ART tips to avoid pipet contamination. 13. Methacrylate 1.5 mL cuvettes 14. 42-degree water bath 15. 12 LB + 50µg/mL Kanamycin plates per lab section 16. 6 sets of SOC media 17. 6 sets of bacteria speaders. 18. 10X buffer for ligation reaction 19. T4 DNA ligase (6 microliters per lab section) 20. Gas burner and cell spreader for plating cells 46 Laboratory #5 Required Supplies and Equipment For TA: Supplies Needed per lab section of 18 students. 6 groups of 3 3. Lysis Buffer Lysis Buffer For 1 L 10 mM HEPES pH 7.5 10 mL of 1 M 50 mM EDTA 100 mL of 0.5 M 0.02 % Sodium Azide 1 mL of 20% Stock 4. Lysozyme and DNAse solutions 5. Liquid nitrogen for cell freezing 6. 1.5 mL tubes racks (6) 7. Bag of 1.5 mL Eppendorf tubes 8. 6 ice buckets with ice 9. 6 sets of micro pipettors (P1000, P200, P20). 7th set of micro pipettors is for TA 10. 6 sets of micropipettor tips (P1000 and P200) Preferably the ART tips to avoid pipet contamination. 11. 3 microcentrifuges (2 new Eppendorf and one old BioRad) 12. Spray bottle with Ethanol 13. Waste containers for tips etc. (6) Laboratory #6 Required Supplies and Equipment For TA: Supplies Needed per lab section of 18 students. 6 groups of 3 1. AKTA Start Chromatography system 2. Chromatography buffers A and B Chromatography Buffer A For 500 mL Chromatography Buffer B For 500 mL 10 mM Hepes 5 ml of 1 M 10 mM HEPES Buffer 5 ml of 1 M 5 mM EDTA 5 ml of 0.5 M 5 mM EDTA 5 ml of 0.5 M No NaCl 0 ml of 5 M 1 M NaCl 100 ml of 5 M 3. Student protein samples from previous week 4. Ice bucket with ice 5. Spray bottle with Ethanol 6. Waste containers for tips etc. (6) 47 Laboratory #7 Required Supplies and Equipment For TA: Supplies Needed per lab section of 18 students. 6 groups of 3 1. Precast 4-20% gels (6, one for each group) 2. Green clamp to pry open the precast gels after they are run 3. Protein sample (6, one for each group) 4. Electrophoresis apparatus (3, 2 groups per apparatus) 5. Sample buffer (6, one for each group) 6. Running buffer (6, one for each group) 7. P20 Pipettor and tips 8. Electrophoresis tank and dam (6, one for each group) 9. Heat block set to 98 ⁰C 10. Spray bottle with Ethanol 11. Waste containers for tips etc. (6) Laboratory #8 Required Supplies and Equipment For TA: Supplies Needed per lab section of 18 students. 6 groups of 3 1. Spray bottle with Ethanol 2. Waste containers for tips etc. (6) 3. BSA standard (aliquoted for each of 6 groups per section). 4. Reagent A and B for the BCA assay (aliquoted for each of 6 groups per section) 5. Cuvettes for the spectrophotometer (2 for each of 6 groups per section) 6. A portable PASCO wireless spectrophotometer (6) 7. Pipettors and tips (6 sets including 6 P10 pipettors) 8. 15 microcentrifuge tubes and a tube rack (6 sets for each of 6 groups) 9. A bottle of deionized water (one per group, 6 total per section) 10. One 50 ml conical tube (6 total per section) Laboratory #9 Required Supplies and Equipment For TA: Supplies Needed per lab section of 18 students. 6 groups of 3 1. Spray bottle with Ethanol 2. Waste containers for tips etc. (6) 3. Portable PASCO wireless spectrophotometers 4. Purified β-Galactosidase enzyme (0.08 mg/ml) 5. ONPG (1 mg/ml (3.3mM)) supplied as a 10 X stock 6. Reaction buffer (10mM HEPES pH 7.5, 50 mM NaCl, 5 mM EDTA, 10mM MgCl2) 7. Pipettors and tips 8. Microcentrifuge tubes and tube racks 9. Spectrophotometer cuvettes (one each for negative control and experimental).