Chapter 4: Culturing, Expression, Lysis And SDS-PAGE .
Protein Expression and Purification SeriesChapter 4: Culturing, Expression, Lysis and SDS-PAGEAnalysis for 11 ml Cultures for Centrifugation PurificationProcessCell Culture and InductionStudent WorkstationsEach student team requires the following items to streak a starter plate to produce single bacterial colonies:Material Needed for Each WorkstationQuantityLB/amp plateSterile inoculating loop2–20 µl adjustable-volume micropipet and tipsMarking pen1111Common WorkstationQuantityRehydrated BL21(DE3) E. coli containing pDHFRIncubator set to 37 C11Each lab team will streak their own starter plate as a source of cells for culture for protein production.LB/amp plates should be streaked for single colonies and incubated at 37 C for 16–24 hours before theinitial culture activity is planned.Student Protocol: Streaking starter plates to produce single bacterial colonieson agar plates1.Pipet 10 µl of reconstituted E. coli using a sterile pipet tip onto an LB/amp plate. Use a sterile loop toperform the streaking. Streaking takes place sequentially in four quadrants. The first streak is to justspread the cells out a little. Go back and forth with the loop about a dozen times in one quadrant asshown in Figure 4.1a. In subsequent quadrants the cells become more and more dilute, increasing thelikelihood of producing single colonies.2.For subsequent streaks, the goal is to use as much of the surface area of the plate as possible. Rotatethe plate approximately 45 degrees (so that the streaking motion is comfortable for your hand) andstart the second streak. Do not dip into the rehydrated bacteria a second time. Go into the previousstreak about two times and then back and forth as shown in Figure 4.1b for a total of about 10 times.3.Rotate the plate again and repeat streaking (Figure 4.1c).4.Rotate the plate for the final time and make the final streak (Figure 4.1d). When you are finishedstreaking the plate, cover it immediately to avoid contamination. Label the plate with your initials.Chapter 4: 11 ml Culture Protocol forCentrifugation Purification91CHAPTER 411 ml CULTUREPROTOCOLUsing the rehydrated BL21(DE3) E. coli containing pDHFR at the Common Workstation to streakone plate. The purpose of streaking is to generate single colonies from a concentrated suspension ofbacteria. A minute amount of the bacterial suspension goes a long way. Under favorable conditions,one cell multiplies to become millions of genetically identical cells in just 24 hours. There are millions ofindividual bacteria in a single 1 mm bacterial colony.
Protein Expression and Purification SeriesFigure 4.1. Streaking a plate to generate single colonies.5.Place the plate upside down inside the incubator overnight at 37 C. Once colonies have grown to1 mm in diameter, they may be stored in a sealed bag upside down at 4ºC until ready to use for up toone month.6.E. coli forms off-white colonies that are uniformly circular with smooth edges. Avoid using plates withcontaminant colonies.CHAPTER 411 ml CULTUREPROTOCOL92Chapter 4: 11 ml Culture Protocolfor Centrifugation Purification
Protein Expression and Purification SeriesOvernight Cell CultureStudent WorkstationsEach student team requires the following items to prepare an overnight cell culture:Material Needed for Each WorkstationQuantityLB/amp plate streaked with BL21(DE3) containing pDHFR50 ml conical tube containing 3 ml sterile LB/amp broth20% sterile glucose20–200 µl adjustable-volume micropipet and tipsMarking pen11300 µl11Common WorkstationQuantityTube roller in 37 C incubator, shaking water bath or shaking incubator at 37 C1Overnight Culture1.Label the 50 ml conical tube containing 3 ml of sterile LB/amp broth with your initials.2.Add 150 µl of 20% sterile glucose to the 3 ml of sterile LB/amp broth to make a final 1% glucose inLB/amp broth solution.3.Pick a single colony from the starter plate using a micropipettor with a sterile pipet tip and inoculatethe 3 ml of 1% glucose, LB/amp broth by twirling the pipet tip vigorously in the broth to dislodge thecells. Recap the tube. Incubate/shake the culture at 37 C for 12–18 hours.CHAPTER 411 ml CULTUREPROTOCOLNote: If using a tube roller in an incubator, make sure to close the cap tightly on the 50 ml conical tubeto avoid leakage.Chapter 4: 11 ml Culture Protocol forCentrifugation Purification93
Protein Expression and Purification SeriesSubculture and InductionStudent WorkstationsEach student team requires the following items to prepare a sample of uninduced cells for SDS-PAGEanalysis, to subculture the cells, and to induce the cells:Material Needed for Each WorkstationQuantity50 ml conical tube containing 3 ml overnight culture50 ml sterile conical tube containing 11 ml sterile LB/amp broth warmed to 37 CLB/amp broth in two 2 ml microcentrifuge tubes for spectroscopyScrewcap microcentrifuge tube, 1.5 ml100 mM IPTGLaemmli sample buffer20–200 µl adjustable-volume micropipet and tips100–1,000 µl adjustable-volume micropipet and tipsSemi-microvolume cuvettesParafilm squaresMarking pen114 ml125 µl1 ml11421Common WorkstationQuantityTube roller in a 37 C incubator, shaking water bath or shaking incubator set to 37 CWater bath or dry bath set to 95 CMicrocentrifuge with variable speed setting 16,000 x gSpectrophotometer1111–2Prepare uninduced SDS-PAGE sample1.Label a 1.5 ml screwcap microcentrifuge tube “Uninduced PAGE” with your initials.2.Aliquot 100 µl of the overnight culture into the tube.3.Centrifuge the tube at 16,000 x g for 2 minutes.4.Use a pipet to gently remove and discard the supernatant without disturbing the pellet.5.Add 100 µl Laemmli sample buffer to the pellet and fully resuspend the pellet by pipeting up and downor vortexing.Note: It is critical to use only clean pipet tips when aliquoting Laemmli sample buffer or all futuresamples can become contaminated.CHAPTER 411 ml CULTUREPROTOCOL6.Heat the tube at 95 C for 5 minutes.7.Store the sample at –20 C until ready to analyze via SDS-PAGE analysis.8.Write your initials on your tube of Laemmli sample buffer and save this tube. This buffer will be usedthroughout the experiment.94Chapter 4: 11 ml Culture Protocolfor Centrifugation Purification
Protein Expression and Purification SeriesMeasure cell density of overnight culture1.Prepare a 1:10 dilution of your overnight culture by combining 900 µl of sterile LB/amp broth with100 µl of overnight culture in a clean semi-microvolume cuvette.2.Cover the cuvette with parafilm and gently invert the cuvette several times to mix the diluted overnightculture.3.If not already set up, set a spectrophotometer to read absorbance at 600 nm and then blank theinstrument using 1 ml LB/amp broth in a cuvette. (Note: Save this cuvette containing LB/amp broth touse as a blank for subsequent readings.) If using a Bio-Rad SmartSpec Plus spectrophotometer, seeAppendix D for instructions on how to use the instrument.4.Measure the absorbance at 600 nm (OD600) of the 1:10 dilution of overnight culture cells.OD600 of 1:10 dilution of overnight culture:OD600 of overnight culture OD600 of 1:10 dilution x 10:Prepare a subculture with a starting OD600 of 0.3Prepare an 11 ml culture with an OD600 of 0.3 by combining the correct volumes of overnight culture andsterile LB/amp broth that has been warmed to 37 C.X ml of overnight culture 1.11 ml x 0.3OD600 of overnight cultureCalculate the amount of overnight culture that you will need to prepare a subculture with an OD600 of0.3 and record the value below.2.Retrieve your 50 ml conical tube containing 11 ml of LB/amp broth that has been warming to 37 Cand label it with your initials.3.Remove and discard a volume of LB/amp broth, equivalent to the volume you calculated in step 1,from the 50 ml conical tube of overnight culture using a micropipetor and sterile tip.4.Gently swirl your overnight culture to resuspend all the cells, then add the volume you calculatedof overnight culture in step 1 to the remaining LB/amp broth in the 50 ml conical tube using amicropipetor and sterile tips.5.Screw the cap of the 50 ml conical tube on tightly and grow the 11 ml culture at 37ºC, with shaking orrotating for 1 hour.6.Record the time that you start your subculture below.Subculture Start Time:7.Record the time you stop your subculture below.Subculture End Time:Chapter 4: 11 ml Culture Protocol forCentrifugation Purification95CHAPTER 411 ml CULTUREPROTOCOLml of overnight culture
Protein Expression and Purification SeriesMeasure OD600 of subculture and induce GST-DHFR-His expression1.After one hour of incubation measure the OD600 of your subculture.2.Blank a spectrophotometer at 600 nm with 1 ml LB/amp broth.3.Prepare a 1:3 dilution of your subculture by combining 600 µl of sterile LB/amp broth with 300 µl ofsubculture in a clean semi-microvolume cuvette. Cover the cuvette with parafilm and invert gentlyseveral times to mix well.4.Measure the OD600 of your 1:3 diluted subcultured cells and record the results below. Next calculatethe OD600 of the subcultured cells and record the value.OD600 of 1:3 dilution of subcultured cells:OD600 of subcultured cells OD600 of 1:3 dilution x 3:5.Add 25 µl of 100 mM IPTG to the subculture in the 50 ml conical tube and continue incubating andshaking the culture at 37ºC for 4–24 hours. During this time period, most of the resources of the E. coliwill go toward producing the recombinant GST-DHFR-His protein rather than cell division, though celldivision does still occur.6.Record the time that you start and end your induction below.Induction Start Time:Induction End Time:CHAPTER 411 ml CULTUREPROTOCOL96Chapter 4: 11 ml Culture Protocolfor Centrifugation Purification
Protein Expression and Purification SeriesCollecting cell pellet and lysing cellsStudent WorkstationsEach student workstation requires the following items to isolate its cell pellet, to prepare an induced cellsample for SDS-PAGE analysis, and to lyse its cells:Material Needed for Each WorkstationQuantityInduced cell cultureLysis buffer 1Lysis buffer 2Microcentrifuge tubes, 2 mlScrewcap microcentrifuge tube, 1.5 mlLaemmli sample buffer (leftover from previous activity)20–200 µl adjustable-volume micropipet and tips100–1,000 µl adjustable-volume micropipet and tipsMarking pen1 ml500 µl500 µl311 ml111Common WorkstationQuantityWater bath or dry bath set to 95 CMicrocentrifuge with variable speed setting 16,000 x gVortexerDry ice/ethanol bath1111Prepare Induced SDS-PAGE Sample1.Label a 1.5 ml screwcap microcentrifuge tube “Induced PAGE” with your initials.2.Aliquot 100 µl of the induced culture into the tube.3.Centrifuge the tube at 16,000 x g for 2 minutes.4.Use a pipet to gently remove and discard the supernatant without disturbing the pellet.5.Add 100 µl Laemmli sample buffer to the pellet and fully resuspend the pellet by pipeting up and downor vortexing.6.Heat the tube at 95 C for 5 minutes.7.Store the sample at –20 C until ready to analyze via SDS-PAGE.1.Label two 2 ml microcentrifuge tubes with “Induced Cell Pellet” with your initials. Pellet the inducedcells by aliquoting 2 ml of induced cell culture into each of the two labeled 2 ml microcentrifuge tubes.Centrifuge the two 2 ml microcentrifuge tubes at 16,000 x g for 2 minutes. Use a pipet to gentlyremove and discard the supernatant from both tubes without disturbing the pellets.2.Aliquot 2 more ml of induced overnight culture into each of the two microcentrifuge tubes labeled“Induced Cell Pellet” and centrifuge at 16,000 x g for 2 minutes. Discard the supernatant. Repeatuntil all of the culture has been centrifuged and each of the two tubes contains a cell pellet. Store thecell pellet at –20 C or continue to lyse the induced cells.Chapter 4: 11 ml Culture Protocol forCentrifugation Purification97CHAPTER 411 ml CULTUREPROTOCOLPelleting induced cells
Protein Expression and Purification SeriesLysing induced cells1.Add 250 µl Lysis buffer 1 to each of the two Induced Cell Pellet tubes. Resuspend the cell pelletsthoroughly by pipeting or vortexing and make sure that there are no cell clumps remaining in both ofthe tubes.Note: As lysis occurs, the solution will get more viscous. Vigorous vortexing and pipetting might benecessary to resuspend the pellet.2.Label a clean 2 ml microcentrifuge tube “Lysed Cells” with your initials. Combine the two 250 µl lysedcell fractions into the labeled microcentrifuge tube for a total of 500 µl of lysed cell solution.Note: Use lab tape for the labeling of the 2 ml microcentrifuge tube since the ethanol in the dry ice/ethanol bath will remove the markings from the tube, but not from the tape.3.Place the tube of lysed cells in the dry ice/ethanol bath for at least five minutes, until the solution iscompletely frozen. Be careful not to allow the dry ice/ethanol to come in contact with your skin! Afterfive minutes, remove the tube from the dry ice/ethanol bath and thaw completely. It is acceptable tothaw in a 37 C waterbath. Do not thaw by hand to avoid freezerburn to your hand.Note: Be careful not to allow the dry ice/ethanol to come in contact with your skin.4.Repeat two more freeze-thaw steps for a total of three freeze-thaw cycles.5.After the final freeze-thaw step, add 500 µl Lysis buffer 2 and thoroughly mix by pipetting or vortexing.6.The lysed cells can be stored at –20 C until they are ready to be purified.Note: Another method that can be used for efficient lysis is freezing the cells in a –70 C freezer. The cellscan be frozen at –70 C overnight and then thawed completely when separation of the soluble fromthe insoluble fraction is to be performed. If neither dry ice/ethanol or a –70 C freezer is availalble, it ispossible to perform two freeze/thaw cycles using a –20 C freezer. Samples can be placed at –20 Covernight (check that samples are completely frozen), thawed the next day and then placed at –20 Covernight again before purification.CHAPTER 411 ml CULTUREPROTOCOL98Chapter 4: 11 ml Culture Protocolfor Centrifugation Purification
Protein Expression and Purification SeriesSeparating Soluble from Insoluble Induced Cell Fractions andPreparing SDS-PAGE SamplesStudent WorkstationsEach student team requires the following items to separate soluble from insoluble lysate fractions andprepare soluble and insoluble fraction samples for SDS-PAGE analysis:Material Needed for Each WorkstationQuantityThawed cell lysateLysis buffer 2Screwcap microcentrifuge tube, 1.5 mlMicrocentrifuge tubes, 2 mlLaemmli sample buffer (left over from the previous activity)20–200 µl adjustable-volume micropipet and tips100–1,000 µl adjustable-volume micropipet and tips10 ml syringe22 gauge syringe needleMarking pen1 ml1 ml231 ml11111Common WorkstationQuantityWater bath or dry bath set to 95 CMicrocentrifuge with variable speed setting 16,000 x g11Separate soluble from insoluble cell lysate components1.Make sure that your lysate is balanced with either another student’s lysate fraction or with amicrocentrifuge tube filled with water.2.Separate the insoluble fraction of the lysed cells from the soluble fraction by centrifugation at16,000 x g for 20 minutes.3.Label a clean 2 ml microcentrifuge tube “Soluble fraction” with your initials. Gently pour thesupernatant from the “Lysed Cells” tube into the “Soluble fraction” tube being very careful not todecant any of the insoluble fraction (the opaque, viscous blob that is mainly composed of genomicDNA) into your soluble fraction.4.Relabel the “Lysed Cells” microcentrifuge tube containing the remaining insoluble fraction “Insolublefraction.”5.Add 1 ml of Lysis buffer 2 to the “Insoluble fraction” tube and resuspend the pellet by shearing witha syringe needle.Note: The insoluble fraction contains a large quantity of genomic DNA that can be quite viscous,making resuspension difficult. Using a 22 gauge needle, the insoluble fraction can be pulled up into a3 ml syringe and expelled from the syringe into a clean tube multiple times to decrease the viscosity ofthe DNA. Ensure that the needle is disposed of properly in a sharps container.6.Remove 50 µl of “Soluble fraction” and place it in a clean 1.5 ml screwcap microcentrifuge tubelabeled “Soluble PAGE” with your initials. Add 50 µl Laemmli sample buffer and mix thoroughly.Chapter 4: 11 ml Culture Protocol forCentrifugation Purification99CHAPTER 411 ml CULTUREPROTOCOLNote: The insoluble fraction does not necessarily adhere to the tube so extreme care is needed whendecanting.
Protein Expression and Purification Series7.Remove 50 µl of “Insoluble fraction” and place it in a clean 1.5 ml screwcap microcentrifuge tubelabeled “Insoluble PAGE” with your initals. Add 50 µl Laemmli sample buffer and mix thoroughly.8.Heat the “Soluble PAGE” and “Insoluble PAGE” samples at 95 C for five minutes.Note: Make sure that you are heating your blue SDS-PAGE samples and not your actualsoluble and insoluble fractions!9.Store the SDS-PAGE samples “Soluble PAGE” and “Insoluble PAGE” at –20 C until ready toanalyze via SDS-PAGE.10. Store the soluble fraction and Insoluble fraction at –20 C until ready to purify the soluble fraction.CHAPTER 411 ml CULTUREPROTOCOL100Chapter 4: 11 ml Culture Protocolfor Centrifugation Purification
Protein Expression and Purification SeriesAffinity and Size Exclusion (Desalting) Purification ofGST-DHFR-HisStudent WorkstationsEach student team requires the following items to purify their GST-DHFR-His samples using affinitychromatography followed by size exclusion chromatography (desalting):Material Needed for Each WorkstationQuantitySoluble lysate fractionProfinity IMAC Ni-charged resin slurryDistilled waterEquilibration bufferWash bufferElution bufferEmpty Micro Bio-Spin column with cap and yellow tip closureDesalting columnMicrocentrifuge tubes, 2 mlScrewcap microcentrifuge tubes, 1.5 mlLaemmli sample buffer (leftover from previous activity)2–20 µl adjustable-volume micropipet and tips20–200 µl adjustable-volume micropipet and tips100–1,000 µl adjustable-volume micropipet and tipsMarking pen600–750 µl250 µl slurry200 µl500 µl600 µl400 µl11641 ml1111Common WorkstationQuantityTube roller or rocking platformWater bath or dry bath set to 95 CMicrocentrifuge with variable speed setting 16,000 x g111Pouring, washing, and equilibrating column1.Snap off the bottom tab of the empty Micro Bio-Spin column, label the column with your initials andplace the column in a clean 2 ml microcentrifuge tube.2.Thoroughly resuspend the resin and pipet 200 µl of the slurry into the column. Centrifuge for 2 minutesat 1,000 x g to remove the packing. (See Appendix C—Instructions on how to calculate the speed thatneeds to be set to generate a relative centrifugal force, RCF, of 1,000 x g.) Discard the buffer that hascollected in the 2 ml microcentrifuge tube. Place the column back into the 2 ml microcentrifuge tube.3.Wash the column by gently adding 200 µl of distilled water to the top of the column taking care notto disturb the resin. Centrifuge for 2 minutes at 1,000 x g to remove the water. Discard the water thatwas collected in the microcentrifuge tube and place the column back into the 2 ml microcentrifugetube.4.Equilibrate the column by gently adding 500 µl of Equilibration buffer and centrifuge for 2 minutes at1,000 x g to remove the Equilibration buffer. Discard the buffer and the 2 ml microcentrifuge tube. Donot discard the column!Chapter 4: 11 ml Culture Protocol forCentrifugation Purification101CHAPTER 411 ml CULTUREPROTOCOLNote: The resin is provided as a 50% slurry in 20% ethanol. It is very important to thoroughlyresuspend the resin before pipeting, or the incorrect quantity of resin will be aliquoted.
Protein Expression and Purification SeriesSample binding to the column1.Attach a yellow tip closure to the bottom of the column.2.Add up to 600 µl of soluble fraction of the lysate to the column and attach the clear top cap onto thecolumn.3.Gently mix the column for 20 minutes at room temperature on a tube rotator or a mini rocker. (Auniform slurry should be formed.)4.Lab
96 Chapter 4: 11 ml Culture Protocol for Centrifugation PuriÞcation CHAPTER 4 11 ml CULTURE PROTOCOL Measure OD600 of subculture and induce GST-DHFR-His expression 1. After one hour of incubation measure the OD600 of your subculture. 2. Blank a spectrophotometer at 600 nm with 1 ml LB/amp broth. 3.
pierce Ip lysis buffer (Thermo Scientific . Lysis buffer C Lysis buffer D Lysis buffer C Lysis buffer D 12 3 4 58691071112 A B Pair #3 Pair #4 D Pair #1 Pair #2 G H. 4 Start-up Guide Micropla
Part One: Heir of Ash Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10 Chapter 11 Chapter 12 Chapter 13 Chapter 14 Chapter 15 Chapter 16 Chapter 17 Chapter 18 Chapter 19 Chapter 20 Chapter 21 Chapter 22 Chapter 23 Chapter 24 Chapter 25 Chapter 26 Chapter 27 Chapter 28 Chapter 29 Chapter 30 .
Activity 1: Make Lysis Buffer 1. Add 5 g of salt (NaCl) to the empty 50 mL lysis buffer tube. 2. Add 45 mL of water (H₂O) and 5 mL of clear liquid detergent to the lysis buffer tube. 3. Place the cap securely on the tube and mix gently by swirling or inverting the tube. 4. You have made a lysis buffer. Activity 2: Make Strawberry Lysate 1.
Cell Lysis: Cell lysis from tissue or cell culture is straightforward for Western blotting applications in which harsh lysis conditions (e.g., an SDS-sample loading buffer) will not interfere with the results, as the sample will be denatured during processing. For an IP/co-IP, the lysis
TO KILL A MOCKINGBIRD. Contents Dedication Epigraph Part One Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10 Chapter 11 Part Two Chapter 12 Chapter 13 Chapter 14 Chapter 15 Chapter 16 Chapter 17 Chapter 18. Chapter 19 Chapter 20 Chapter 21 Chapter 22 Chapter 23 Chapter 24 Chapter 25 Chapter 26
DEDICATION PART ONE Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10 Chapter 11 PART TWO Chapter 12 Chapter 13 Chapter 14 Chapter 15 Chapter 16 Chapter 17 Chapter 18 Chapter 19 Chapter 20 Chapter 21 Chapter 22 Chapter 23 .
(PCI) Phenol:Chloroform:Isoamyl alcohol (25:24:1) (CI)Chloroform:Isoamyl alcohol (24:1) Sodium Acetate (NaAc) 3M Isopropanol 100% Ethanol 70% DNA Extraction steps 1m 1 Each tube of lysis buffer will be split in half so prepare one tube of lysis buffer for two samples. Prepare Lysis Buffer by adding to each 2mL microcentrifuge tube 650 µl µL .
Relying on pen and ink, which is the conventional medium used for archaeological illustration (Dillon, B. 1987), the artist was able to use semiotic principles to turn traditional scientific illustrations into allegories of the subjective experience (figure 4). Figure 4 Illustrations have been used to draw the viewers’ attention to the skill of making and the focal points of the fledgling .
