or A. thaliana). After cell lysis is complete, centrifugation, the Bradford assay, and possibly

Transcription

1 BIOSC147: Lab 3c Use of protein extraction, purification and quantitation of with spectrophotometry and standard curves to investigate protein expression, structure, and function. Cell lysis to generate protein extracts from harvested cells (S. cerevicae, C.elegans, C. reinhartii or A. thaliana). Separation of cell debris from extracts. Protein quantitation assay using standard curve. INTRODUCTION and BACKGROUND REFERENCES: To purify protein, it must be released from cells, kept stable, separated from contaminants, and measured accurately. Chemical and physical properties of biological material, molecules and lab solutions play a major role in these steps. In preparing for and analyzing data from this lab you must correctly perform calculations for, prepare buffers, and carry out clear calculations on data you ve recorded accurately. Your ability to make useful contributions to your team depends on your careful analysis of previous lab results, understanding of chemical properties of biological molecules and buffers, enzyme activity, protein expression and cell organization in your model organism, and appropriate calculations for setting up experiments and recording and reporting results. To prepare, gather relevant information from lecture, Chapters 2, 3, 5, 6, and Chapter 8. Chapter 18. and parts of Chapters 26, 27, 28, 30, 31, 32 are still good references. Be sure to complete your analysis of results from lab 3a and lab 3b so you can a) get your new lab partner up to speed b) be able to set up your enzyme digest and c) be able to perform calculations accurately as you analyze the results. Review lab 2 and lab 1c to remember important techniques. QUESTION and OBJECTIVE(S): In this lab you will investigate which conditions are best for lysing cells and extracting protein from your cultured cells. Along with the enzyme treatment identified in lab 3a, we will also disrupt cultured cells with a variety of physical and chemical techniques to extract protein from your various GFP-expressing organisms (S cerevicae, C elegans, C. reinhartii, or A. thaliana). After cell lysis is complete, centrifugation, the Bradford assay, and possibly fluorescence measurements will be used to purify and analyze your extracts. On the first day, you will innoculate cultures of the organism you studied in lab 3a and learn assay techniques. On the second day, you will measure growth using spectrophotometry and harvest cells using centrifugation to separate culture media from cells. In preparation for quantitative analysis of protein extraction, you will weigh the harvested pellets. The purpose of this lab is to continue the process of analyzing proteins using chromatography, SDS-PAGE, ELISA technologies, and determination protein concentration with a microtiter plate format assay. You will also improve your ability to identify and define the structure, function, and organismal distribution of carbohydrates, lipids, proteins, and nucleic acids during this week s lab. PROCEDURE: - You will be in new groups of 4, each member brings a different cultured organism sample to the experiment from the culture in lab 3b. - GENERAL ORGANIZATION: o You and your teammates will choose a combination of extraction buffers, physical disruption method, and use of enzyme. You have two goals: One is to successfully extract soluble protein from the culture for use in lab 3d. The other is to scientifically compare methodology to determine which method is better for extracting protein. o With your partner, choose two combinations of buffers, physical disruption, and use of enzyme that you would like to compare for the organism(s) you will be using. o ON the lab day, discuss your choices with the other members of your lab group, and adjust protocols to make sure the work done by all 4 of you is scientifically valid. o Each team will test extraction procedures on multiple organisms, and you will also share data with members of your 3a, 3b, and 3c teams to identify the best procedure for each organism. BIOSC147_Lab3c.docx Page 1 of 7 August 12, 2016

2 - BUFFER PREPARATION: o Prepare reagents, label supplies, and organize a workstation based on the extraction procedure your group has chosen. o Choose from 5 possible buffers, with the option for varying ph. All working buffers should contain protease inhibitor cocktail at 1X concentration. Buffers differ in composition by salt identity and concentration, glycerol concentration, type and concentration of detergent, chelator, and buffer identity. Here is a list of the buffers you can make.! HEPES ph ! TRIS ph ! Hypotonic Lysis Buffer (by Chris Bell)! Plant nuclear extraction buffer (a la Harley Smith)! New better buffer (Harley Smith/Katherine Krolikowski) o Stocks to be used as buffer ingredients will be available. Choose an appropriate volume to make. Note: for lab 3d, one sample run will use 2mL maximum extract. Extractions should be performed so that the extract contains the highest concentration of protein possible. Usually, a 1:1 ratio of tissue to extraction buffer is a good starting point, and small amounts of buffer can be added to reach a viscosity of the solution for reasonable manipulation using micropipettes. Use small flasks and careful to prepare you chosen buffers. Here are stock solutions that will be available o BUFFER RECIPES: Here are recipes for various buffers that can be used for protein extraction from tissues and cells. Use these as references to prepare an appropriate amount for your use. Remember that you may include enzymes (same stocks as in lab 3a in these buffers, either before or after the physical disruption step). Prepare your buffers in small flasks using concentrated stocks and accurate metrology. BIOSC147_Lab3c.docx Page 2 of 7 August 12, 2016

3 - EXTRACTION PROCEDURES: o Each procedure, combines enzymatic treatment with some type of physical disruption. Review the results of lab 3a to determine the best enzyme treatment to use for your organism (this may be different than the enzyme treatment your teammates choose, as they bring different organsims) o Before you begin the extraction, record the mass of your tissue/cell starting material. You will report your results in terms of protein extracted per gram of tissue. o Extracts MUST be run through a 0.45um filter before they can be injected into the FPLC in lab 3d. It is better to filter your extracts during lab 3c, if time allows. BIOSC147_Lab3c.docx Page 3 of 7 August 12, 2016

4 o FREEZE-THAW (ice crystal) METHOD: from - Cell pellets should be in round base tubes, no more than 0.8 g of wet weight cell pellet per tube - If cell pellet is frozen, thaw in a mixture of ice and water. - Dissolve pellet in 200 µl of ice cold extraction buffer with inhibitors. - Add 700 µl volume of acid washed glass beads. - Vortex sample at maximum speed for 30 sec. - Important: Efficient breakage is visible by the sample forming a whitish film and tiny bubbles on the inside wall of the tube - Place sample in a mixture of ice and water for 30 sec. - Repeat the vortex and ice water steps seven more times. - Tip: If more than one sample needs to be processed the time spent for cell breakage can be shortened by vortexing 2 samples at a time on 2 vortexers for 30 sec. While these samples rest for 30 sec the next 2 samples can be vortexed. - Spin samples at 4 C for 3 min at low g (e.g. 2,500g). If a centrifuge for round base tubes is not available, transfer samples to microfuge tubes and spin in microfuge centrifuge at low speed. - Transfer supernatant to fresh microfuge tubes. - Spin in microfuge centrifuge at 4 C for 10 min at 10,000 rpm. - Transfer supernatants to fresh tubes. - Note: A highly concentrated WCE has an opaque appearance. A yellowish colour is due to pigments and does not necessarily indicate good breakage. HYPOTONIC LYSIS METHOD (From Chris Bell, PhD) - Obtain your frozen tissue/cell pellet from the -20 freezer. - Place the tube containing the pellet in your ice bucket. Keep tubes on ice at all time for remaining steps - Immediately add hypotonic lysis buffer (2:1 ratio of buffer to pellet) to the tube containing your pellet. Do not allow the pellet to thaw before lysis buffer is added - Vortex the pellet until it is dispersed into the lysis buffer. Place the tube back on ice. - Incubate on ice for 15 minutes - Transfer contents of tube to microcentrifuge tube(s) - Spin the tube in the microfuge at 14K for 3 minutes. - Carefully transfer the supernatant to a newly-labeled fresh tube. Take care not to disturb the pellet - Dispose of the pipette tip and the tube with the pellet in biohazard waste BIOSC147_Lab3c.docx Page 4 of 7 August 12, 2016

5 MORTAR AND PESTLE METHOD - Quickly harvest ~1-2g of tissue/cells directly into mortar. Mortar can be frozen (minus 80 C) or chilled at 4 C. - Grind tissue or cells to a fine powder using mortar and pestle. Vigorous grinding is necessary, and sea sand may be added to increase disruption, if desired. - Add extraction buffer (1:1 ratio with tissue/cells) o Use as little buffer as possible that allows all tissue to go into solution o Add buffer in 250uL increments - If frozen, let ground tissue/cells thaw into extraction buffer - Note total volume of buffer used - Disrupted tissue/cells are now suspended in extraction buffer - Using a p1000 micropipette, transfer liquid to a microfuge tube - Spin in microcentrifuge at high speed to pellet cell debris - Carefully transfer the supernatant to a newly-labeled fresh tube. Take care not to disturb the pellet - Dispose of the pipette tip and the tube with the pellet in biohazard waste DOUNCE METHOD - Remove pelleted tissue/cells from -20 C - Add extraction buffer to the pellet, starting with a 1:1 ratio of buffer:pellet. - Cap the tube and vortex to re-suspend the pellet. Add additional buffer as necessary - Pour the solution into the dounce (40mL, 7mL and 2mL sizes available). BE CAREFUL! Dounce is fragile & expensive - Shear (lyse) the cells by slowly moving the dounce all the way to the bottom and back again for a total of three times. - Transfer the sheared cell solution into microcentrifuge tube(s) and close the cap - Place tubes on ice - Rinse the dounce (CAREFULLY) in dh 2 0 three times (fill with water, dump out, repeat). Return dounce (CAREFULLY!) to the nest of paper to dry and for storage. Keep the dounce parts in pairs - Centrifuge the tube 2min at 14K RPM. Be sure the microfuge is balanced - Carefully transfer the supernatant to a newly-labeled fresh tube. Take care not to disturb the pellet - Dispose of the pipette tip and the tube with the pellet in biohazard waste BIOSC147_Lab3c.docx Page 5 of 7 August 12, 2016

6 - GENERAL PROCEDURAL REMINDERS: - ENZYME TREATMENT: Using your and your partners data from lab 3a, decide which (if any) enzymes might improve tissue and cellular disruption. - CENTRIFUGE and FILTRATION o No matter what extraction procedure is used, the last step should be centrifugation to remove solids. o Place your extract in an appropriately-sized container, and centrifuge at high speed, taking care to balance the centrifuge. o Transfer the supernatant to a new container, taking care to leave ALL of the solids (pellet) behind. Accurately measure the volume of supernatant you have and record this. o Filtration using a 0.45um syringe filter will be necessary to remove any other contaminants before loading sample into FPLC. This can be done during lab 3c or 3d. - MEASURING PROTEIN YEILD o Use the Coomassie stain and BSA standards as in previous labs to measure the concentration of protein in your extract using the Bradford assay. 1-2mL cuvette or a 96- well plate format will be available o If available, submit a small (100uL) sample to the 96-well Tecan fluorometry assay to measure GFP content. ANALYSIS NOTES: - Your analysis should include calculations of grams of protein extracted per grams of cultured cells/tissue harvested. You are required to show data and calculations comparing at least 2 different extractions of at least 2 different organisms is required. o g-protein per culture o g-protein per g-tissue/cells o GFP units per g-protein - Growth conditions for each organism should be described - If data is available, report GFP units per gram of protein - Create graphs that illustrate comparisons between different extraction procedures, different culture conditions and between different organisms. - Visit with members of other groups to gather data for use in your analysis. Your final report should include comparisons of all 4 organisms, and for at least 2 organisms, comparisons between different strains of the same organism. - USE SCIENTIFIC REASONING and CLEAR WRITING to draw conclusions about the effectiveness of using of different techniques to both culture organisms and prepare protein extractions - CONCLUSION should include a ranking of which of your group s sample(s) are the best candidates for protein chromatography, with clear reasoning. An explanation of what GFPtagged protein is expected in your extract should be included in this reasoning. BIOSC147_Lab3c.docx Page 6 of 7 August 12, 2016

7 REFERENCES:! - Mortar and pestle without sea sand! Hypotonic lysis buffer! Cracking buffer - ication/cell_lysates_yeast/! Vortexing with glass beads! Dounce (glass or plastic) BIOSC147_Lab3c.docx Page 7 of 7 August 12, 2016