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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?

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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? 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 shak- ing 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 possi- ble. 16 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 pel- let 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.
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