Green Fluorescent Protein (GFP) Purification Kit How Can Proteins Be Purified? What Is the Mechanism Used in Biotechnology to Extract Proteins of Interest?

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Green fluorescent protein (GFP) purification kit How can proteins be purified? What is the mechanism used in biotechnology to extract proteins of interest?

This lesson is a continuation of the pGLO Transformation kit. Students remove a colony of transformed bacteria that results from that lab and treat it to remove and purify the green fluorescent protein (GFP) that it produces. Protein such as insulin, can be created by bacteria in labs, purified, then used as medicine. This lab uses one technique, hydrophobic interaction column (HIC) chromatography to separate GFP from the bacteria that produced it. GFP can be used as a model protein to show that purification. Students begin by using one colony of transformed bacteria from each condition in the pGLO transformation lab. The colonies are added to nutrients to grow, then incubated. After 24-48 hours, the bacteria is centrifuged, resuspended, then an enzyme is added to lyse the bacterial membranes. The samples are frozen to complete the breakdown of the bacteria, then centrifuged again to remove the bacterial debris. The remaining protein samples are then added to the chromatography columns which contain a “bed” of microbeads, these columns are treated with buffers of high salt concentrations that retain the hydrophobic (water-hating) protein (GFP). The final treatment rinses the protein from the column. Reflection 1. Using the data table from Purification Phase 3, compare how your predictions matched up with your observations for each buffer. a. Binding buffer

b. Wash buffer

c. Elution buffer

2. Based on your results, explain the roles or functions of these buffers. Hint: how does the name of the buffer relate to its function. a. Equilibration buffer

b. Binding buffer

c. Wash buffer

d. TE (elution buffer)

3. Were you successful in isolating and purifying GFP from the cloned bacterial cells? Identify the evidence you have to support your answer.

seed2stem.org GFP Purification—Quick Guide

Lesson 2 InoculationGFP Purification—Quick Guide Growing Cell Cultures Lesson 2 Inoculation Growing1. Remove Cell the Cultures transformation plates from the incubator and examine using the UV light. Identify severalGFP green Purification—Quick colonies that Guide 1. areRemove not touching the transformation other colonies plates on from the LB/amp/arathe incubator plate. and examineIdentify usingseveral the white UV Lessoncolonieslight. 2 Identify Inoculation on the several LB/amp green plate. colonies that Inoculation:Growing aregrowing not Cell touching Cultures cell other cultures colonies on the LB/amp/ara LB/amp LB/amp/ara plate. Identify several white 1. Remove thecolonies transformation on the LB/amp plates plate. from the incubator1. and Remove examine the transformation using the UV plates light. from LB/amp/ara LB/amp Identify severalthe incubator green colonies and examine that using are notthe UV touching otherlight. colonies Identify several on the green LB/amp/ colonies that ara plate. Identifyare not severaltouching white other coloniescolonies on the the LB/ampLB/amp/ara plate. plate. Identify several white colonies on the LB/amp plate. LB/amp/ara LB/amp 2. Obtain two culture tubes containing the growth media LB/amp/ara. Label one "+" and one "-". Using a sterile loop, lightly 2. touchObtain the two loop culture to atubes green containing colony andthe 2. Obtain twoimmersegrowth culture media ittubes in LB/amp/ara.the containing "+" tube. Label Using the one a new "+" growth mediasterileand LB/amp/ara.one loop, "-". repeat Using Labelfor a sterile a white one loop, colony "+" andlightly and one "-". Usingimmersetouch a sterile the it inloop loop,the "-"to lightly atube green (it touchis colony very impor- and the loop to tantimmersea green to pick itcolony onlyin the a single"+"and tube. immerse colony). Using Spin a new the it in the "+" tube.loopsterile between Usingloop, repeat a newyour for sterile indexa white loop,finger colony andand immerse it in the "-" tube (it is very impor- repeat for2. a thumbObtain white totwo colony disperse culture and the tubes immerseentire containing colony. it the growthtant to pick media only LB/amp/ara. a single colony). Label Spinone "+"the in the "-" tube (it is very important to pick andloop one between "-". Using your a sterileindex loop,finger lightly and only a single colony). Spin the loop between touchthumb theto disperse loop to the a greenentire colony.colony and LB/amp/ara your index fingerimmerse andit in thumb the "+" to tube. disperse Using a new the entire colony. + LB/amp sterile loop, repeat for a white colony and - immerse it in the "-" tube (it is very impor- LB/amp/ara tant to pick only a single colony). Spin the + LB/amp loop between your index finger and - thumb to disperse the entire colony. 3. Cap the tubes and place them in the shaking incubator or on the shaking platform and culture overnight at 32 °C or 2 days LB/amp/ara 3. Cap the tubes and place them in the shak- 3. Cap the tubesat room and temperature. place them in the shaking LB/amp ing incubator or on the shaking platform + + incubator or on the shaking platform and Incubate at 32 °C overnight - and culture overnightor at 32 °C or 2 days culture overnight at 32 °C or 2 days at or at room temperature. � room temperature.Cap the tubes and shake vigorously by + 2 days at room - Incubate at 32 °C overnight or hand. Place in the incubatoror horizontally temperature or Cap the tubesat 32 and °C shakefor 24–48 vigorously hours. Remove by and � 3. Capshake thethe by tubestubes hand and andperiodically place shake them vigorously when in the possi- shak- by 2 days at room hand. Place in the incubator horizontally at - ble.inghand. incubator Place in or the on incubator the shaking horizontally platform temperature 32 °C for 24–48andat 32 culture °C hours. for overnight 24–48 Remove hours. at and32 Remove°C shake or 2 days and by hand periodicallyatshake room by temperature. hand when periodically possible. when possi- 16 + ble. Incubate at 32 °C overnight or � or Cap the tubes and shake vigorously by 2 days at room

16 - hand. Place in the incubator horizontally temperature at 32 °C for 24–48 hours. Remove and shake by hand periodically when possible.

seed2stem.org Purification Lessonphase 3 Purification1: Phase 1 bacterial concentrationBacterial Concentration 1. Label one microtube1. Label one "+" withmicrotube your "+"name with and your class name period. Removeand your class liquid period. cultures Remove from your the liquid cul- 2 ml shaker and observetures fromwith thethe shaker UV light. and observeNote any with the UV light. Note any color differences color differences between the two cultures. Using between the two cultures. Using a new a new pipette, pipette,transfer transfer 2 ml of 2 "+"ml liquidof "+" cultureliquid culture into the “+” microtube.into the “+” Spin microtube. the microtube Spin the for microtube 5 minutes in the centrifugefor 5 minutes at in maximum the centrifuge speed. at maximum The pipette used inspeed. this step The canpipette be usedrepeatedly in this step rinsed can be � in a beaker of waterrepeatedly and rinsedused forin a all beaker following of water and steps of this laboratoryused for all period. following steps of this laborato- ++ ry period.

2. Pour out the supernatant and observe the pellet + under UV light.2. Pour out the supernatant and observe the pellet under UV light.

3. Using a rinsed3. Usingpipette, a rinsed add 250 pipette, μl of add TE 250solution µl of TE to the tube. Resuspend the pellet thoroughly by solution to the tube. Resuspend the pellet 250 µl TE rapidly pipettingthoroughly up and bydown rapidly several pipetting times. up and down several times.

4. Using a rinsed pipette, add 1 drop of lysozyme to the resuspended bacterial pellet to initiate 4. Using a rinsed pipette, add 1 drop of 1 drop lysozyme enzymatic digestionlysozyme of theto the bacterial resuspended cell wall.bacterial Mix pel- the contents gentlylet to initiate by flicking enzymatic the digestiontube. Observe of the bac- the tube underterial the UVcell light.wall. Mix the contents gently by flicking the tube. Observe the tube under the UV light. � Freezer

5. Place the microtube in the freezer until the next laboratory period. The freezing causes the bacteria to rupture completely. 5. Place the microtube in the freezer until the next laboratory period. The freezing causes the bacteria to rupture completely.

17 seed2stem.org Purification phaseLesson 2: bacterial 4 Purification lysis Phase 2 Bacterial Lysis 1. Remove the microtube from the freezer and 1. Remove the microtube from the freezer thaw using hand warmth.and Placethaw using the tube hand warmth. Place the in the centrifuge and pellettube in the the centrifugeinsoluble and pellet the insol- Thaw � bacterial debris by spinninguble bacterial for 10 minutesdebris by spinning for 10 at maximum speed. minutes at maximum speed. Centrifuge

2. While your tube is spinning, prepare the chromatography column. Remove the cap 2. While your tube is spinning, prepare the and snap off the bottom from the prefilled chromatography column.HIC Remove column. the Allow cap alland of the liquid snap off the bottom frombuffer the to drainprefilled from HICthe column (~3–5 column. Allow all of theminutes). liquid buffer to drain from the column (~3–5 minutes).

3. Prepare the column by adding 2 ml of Equilibration buffer (2 ml) Equilibration Buffer to the top of the col- 3. Prepare the column by adding 2 ml of umn. This is done by adding two 1 ml Equilibration Buffer toaliquots the top with of thea rinsed column. pipette. Drain the This is done by addingbuffer two 1 toml the aliquots 1 ml mark with on a the column. rinsed pipette. Drain theCap buffer the top andto the bottom 1 ml and mark store the col- on the column. Cap theumn top at and room bottom temperature and store until the next 1 ml the column at room temperaturelaboratory period. until the next laboratory period.

4. After the 10 minute spin, immediately remove your tube from the centrifuge. Examine the tube with the UV light. 250 µl 4. After the 10 minute spin,Using immediately a new pipette, remove transfer 250 µl of the your tube from the centrifuge."+" supernatant Examine into the a new microtube tube with the UV light.labeled Using "+". a new Again, pipette, rinse the pipette well transfer 250 μl of the for"+" thesupernatant rest of the steps into ofa newthis lab period. microtube labeled "+". Again, rinse the pipette + + well for the rest of the steps of this lab period.

250 µl

5. Using a well rinsed pipette, transfer 250 µl of binding buffer to the "+" supernatant. 5. Using a well rinsed pipette,Place thetransfer tube in 250 the refrigerator μl of until the binding buffer to the "+"next supernatant. laboratory period. Place the tube in the refrigerator until the next laboratory period. 18

seed2stem.org Equilibration buffer (used in preparation of the column): raises the salt concentration of the column to match the GFP lysate Binding buffer: raises the salt concentration of GFP to cause a conformational change in GFP (changeLesson in the5 Purification tertiary structure Phase 3 of the Lesson 5 LessonPurification 5 Purification Phase 3 Phase 3 protein), exposing the hydrophobic Proteinregion Chromatographyof GFP Protein ChromatographyProtein Chromatography Wash buffer: washes away less hydrophobic,1. Label 3 contaminatingcollection tubes 1–3proteins and place the 1. Label 31. collection Label 3 collectiontubes 1–3 tubesand place 1–3 andthe place the from the column tubes in the foam rack or in a rack supplied tubes in thetubes foamin in your rackthe foamlaboratory.or in racka rack or Removesupplied in a rack the supplied caps from TE (elution) buffer: removesin your GFP laboratory.in from yourthe the laboratory.top Remove column and bottom the Remove caps of the from the column caps fromand place the top andthe bottom topthe and column of bottomthe column in ofcollection the and column place tube and 1. Whenplace the Collection Predictionthe columnthe in column lastcollection ofObservations the in collectionbuffertube 1. has When tubereachedunder the1. When the surface the of tube number last of the lastbuffer ofthe thehas HICUV bufferreached lightmatrix has the(column reachedproceed surface theand toof surfacethe next of step the HIC matrixthe HIC proceed matrix toproceed the next to stepthe next step 1 below.collection tube) 1 1 below. below. Tube 1 (250 µl) + supernatant Sample in 2. Using a new pipette, carefully and gently(250 µl) + supernatant(250 µl) + supernatant binding buffer 2. Using a2. new Using pipette,load a new 250 carefully pipette,µl of the andcarefully “+” gently supernatant and gently onto the Tube 2 load 250 µlload of topthe250 “+” ofµl ofthe supernatant the column. “+” supernatant onto Hold the the onto pipette the tip Sample with top of thetop column. ofagainst the Holdcolumn. the sidethe Holdpipetteof the the column tip pipette wall, tip just against theagainst side of the the side column of the wall, column just wall, just wash buffer above the upper surface of the matrix and let above the upperabovethe surfacethe supernatant upper of surfacethe matrixdrip ofdown theand matrixthe let side and of thelet col- Tube 3 the supernatantthe supernatantumn drip downwall. Examinedripthe side down of the thethe column sidecol- of theusing col- a UV + Sample with umn wall. umnExamine light.wall. theNoteExamine column your the observations. using column a UV using After a UV it stops+ + elution buffer light. Notelight. yourdripping Noteobservations. your transfer observations. After the itcolumn stops After to it collectionstops dripping transferdrippingtube the 2.transfer column the to column collection to collection tube 2. tube 2. 1 Purification phase 3: protein chromatography 1 1 Wash buffer (250 µl) 1. Label 3 collection tubes 1–3 and place the tubes in the foam Wash bufferWash (250 buffer µl) (250 µl) rack or in a rack supplied in your laboratory. Remove the caps from the top and bottom of the3. column Using theand rinsed place pipette, the column add 250 µl of wash in collection tube 1.3. When Using thethe3. Usingrinsedlastbuffer of thepipette, the rinsed and buffer add let pipette, the 250 entirehas µl add ofreached volumewash 250 µl flowofthe wash into the surface of the HIC matrixbuffer proceed and bufferlet thecolumn. to andentire the let volumeExaminethenext entire step flow thevolume below. intocolumn the flow using into the UV column. Examinecolumn.light. theExamine Note column your the using observations. column the UVusing After the UV the col- 2. Using a new pipette, carefully and gently load 250 μl of the light. Notelight. yourumn Noteobservations. stops your dripping, observations. After transfer the col- After it to the tube col- 3. “+” supernatant onto umnthe topstops ofumn dripping, the stops column. transferdripping, Hold it transferto thetube pipette 3. it to tube 3. tip against the side of the column wall, just above the upper 2 surface of the matrix and let the supernatant drip down the side 2 2 of the column wall. Examine the column using a UV light. Note TE buffer (750 µl) your observations. After it stops dripping transfer the column to TE buffer (750TE buffer µl) (750 µl) collection tube 2. 4. Using the rinsed pipette, add 750 µl of TE 3. Using the rinsed pipette, add 250 l of wash buffer and let the 4. Using the4. UsingrinsedBufferμ thepipette, rinsed and add let pipette, the750 entire µl add of 750TEvolume µl of flow TE into entire volume flow intoBuffer the andcolumn.Buffer let thethe and column.Examineentire let thevolume Examine theentire columnflow volume the into column using flow usinginto the the UV light. Note yourthe observations.column.the Examine column.UV light.After theExamine Note columnthe your columnthe using observations.column thestops using the dripping, transfer it toUV tube light. 3. NoteUV light. your Noteobservations. your observations. 4. Using the rinsed pipette, add 750 μl of TE Buffer and let the entire volume flow into the column. Examine the column using 3 3 3 the UV light. Note your observations. 5. Examine all three collection tubes and note 5. Examine5. allExamine three collection all three collectiontubes and tubesnote and note 5. Examine all three collection tubesany and differences note any indifferences color between in the tubes. 1 2 3 any differencesany differences in color between in color the between tubes. the tubes. color between the tubes. ParafilmParafilm or Saran or SaranWrap Wrapthe tubes the tubes and and 1 place 21 3 2 3 Parafilm orParafilm Saran Wrap or Saran the tubesWrap andthe tubesplace and place place in the refrigerator until the nextin the laboratory refrigerator period.until the next laboratory in the refrigeratorin theperiod. refrigerator until the next until laboratory the next laboratory period. period.

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