NOTE ACRYLAMIDE IS NEUROTOXIN YOU MUST WEAR GLOVES.
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1 GST Purfication and Pulldown Part I Instructor: David Deitcher TA: Kristy Lawton In order to study the function of a protein it is often useful to have that protein purified away from others in the cell. One common approach is to fuse your protein to another one that easily purified from E. coli. In this lab you will be working with SNAP-25 fused to Glutathione S-transferase (GST). GST fusions are very convenient because you can simply incubate glutathione beads with a complex mixture of proteins and purify the GST fusion by simply spinning and washing the beads. Once the protein is purified it may be used in a variety of ways. It can be used as an antigen to raise antibodies. It can also be used to test if other proteins bind to it in a GST pulldown assay. Bacteria containing a plasmid with SNAP-25 fused to GST will be growing from the night before. Early in the morning these overnight culture will be diluted and grown in the presence of IPTG, an inducer of the lac operon. Since the promoter of GST-SNAP- 25 fusion gene is under lac control this induces a lot of protein to be made. After 2 hours of induction, the bacteria will be spun down. The resulting bacteria pellet will be lysed and the supernatant will be incubated with glutathione beads to purify the protein. The crude of uninduced bacteria, the induced bacteria, and purified supernatant of the induced bacteria will be separated by SDS-PAGE. Half the gel will be stained with Coomassie Blue which will stain all protein bands and the other half will be blotted and probed with anti-snap-25 antibodies. Procedure Pouring the gel. 1) Assemble the gel plates. Clamp into pouring stand. Take comb and insert. Make a line with a sharpie about 1.0 cm down from the bottom of the comb. 2) Prepare 10% Acrylamide Resolving gel NOTE ACRYLAMIDE IS NEUROTOXIN YOU MUST WEAR GLOVES. Use a disposable tube and add the following components. solution amount in mls water % acrylamide M Tris ph % SDS 0.1 MIX then add 10% ammonium 0.1 persulfate, MIX TEMED MIX
2 Pipet the solution in between the glass plates up to the level of the mark you made, avoid getting bubbles. Then add ~0.5 ml of n-butanol on top of the gel by pipeting the liquid very slowly while moving across the top of the gel so you get a nice even alcohol layer. Let gel polymerize for about 30 minutes. 3) You should see three layers in your gel. The gel itself, water excreted by the gel during polymerization, and alcohol on top. Pour the small amount of alcohol into the sink, then rinse with deionized water about 10 times to get rid of the alcohol then drain gel of all water and blot remaining water away gently. 4) Prepare 5% stacker gel: solution amount in mls water % acrylamide M Tris ph % SDS 0.03 MIX then add 10% ammonium 0.03 persulfate, MIX TEMED MIX Pipet immediately on top of resolving gel until the lower plate is reached. Insert comb without getting bubbles. Let polymerize minutes. Preparing your protein samples: 1) Pipet 1.5 mls of bacteria (both induced and uninduced) into separate tubes. Spin 1 minute. Remove supernatant and freeze pellets. 2) Prepare 1 ml of EasyLyse reagent according to the attached sheet for Protocol for Lysis of Bacterial Culture. 3) Prepare Glutathione beads by mixing the beads to a uniform consistency, then pipet 100 µl into a tube. Add 1 ml of PBS/Tween, mix, spin 15 seconds, remove supernatant, wash beads 3 more times. Remove most of PBS/Tween leave about 50 µl of liquid. 4) Remove bacteria from freezer, add 200 µl of lysis buffer to each tube. Resuspend pellet by pipeting. Let sit 5 minutes. Spin 2 minutes. Transfer supernatants to new tubes. For the uninduced lysate, transfer 25 µl of the solution to a tube containing 25 µl of loading buffer and boil for 3 minutes (this is your uninduced crude ). From the induced lysate, remove 25 µl and pipet into separate tube containing 25 µl of loading buffer and boil for 3 minutes (this is your induced crude ).
3 5) To the remaining 175 µl of the induced lysate, add 300 µl of PBS/Tween. Then using a cut off tip, pipet glutathione beads into the tube. Put on rotator for 30 minutes at room temp. Spin down tube for 15 seconds, remove supernatant, and wash with 500 µl of PBS/Tween resuspend pellet of beads, spin. Repeat twice more. Then remove all supernatant and add 50 µl of loading buffer to the beads. Heat at 95C for 3 minutes. Spin 3 minutes. Transfer 25 µl from the tube to a fresh tube, avoiding the beads. Add 25 µl of loading buffer and boil for 3 minutes (this is your bead purified ). 6) Set up gel for loading. Load 25 µl of each sample in the following order: Molecular Weight marker (MW) Uninduced crude Induced crude Bead purified MW Unindu crude Induced crude Bead purified 4) Run gel at 180 volts until bromophenol blue nears the bottom. Remove gels from apparatus, cut off stacker of gel with a razor blade, cut gel in half, transfer half to dish containing western transfer buffer and the other half to dish with Coomassie Stain. Put Coomassie stained gel on shaker. Preparing the gel for blotting: 1) Wet a piece of PVDF membrane (brand name Immobilon P) (a hydrophobic membrane that you will blot your proteins on) in methanol for 15 seconds. 2) Transfer to deionized water, push membrane under surface with a Millipore forceps until it sinks. Incubate 2 more minutes. 3) Transfer to dish containing western transfer buffer. 4) Add sponges, regular gel blotting paper to western buffer. Insert cassette into dish so that black side is on the bottom. Place sponge as first layer, then regular blotting paper, then gel, then PVDF membrane, then regular blotting paper, then sponge. Avoid bubbles. Close cassette. 5) Place frozen coolant into tank and cassette holder/electrodes. Pour western buffer into tank, insert cassette so black portion of cassette faces black side of electrode (so protein will migrate into PVDF membrane instead of into the bath). Fill up with buffer to top. Close lid and blot at 100V for 1 hour. 6) Remove cassette, carefully take apart layers. Remove PVDF membrane and place protein side up into plastic box containing PBS, 0.05% Tween, plus 5% nonfat dry milk until next week.
4 EasyLyse Bacterial Protein Extraction Solution Cat. No. RP03750 The EasyLyse Bacterial Protein Extraction Solution is designed for lysing bacterial cells for the isolation of proteins, especially recombinant gene products expressed in E. coli, without significant loss of enzymatic activity. 1 EasyLyse Bacterial Protein Extraction Solution is formulated for ease of use as a homogeneous reagent in high-throughput applications. Unlike mechanical methods such as sonication, the enzymatic method utilized by EasyLyse Bacterial Protein Extraction Solution does not generate heat that could denature the protein of interest. Furthermore, by adjusting reaction conditions, users can lyse any amount of cells, from a single colony to several grams. EasyLyse Bacterial Protein Extraction Solution includes a stable non-avian, non-mammalian lytic enzyme that has a high specific activity. This high specific activity allows the addition of less enzyme, minimizing the amount of extraneous protein. The presence of a nuclease in the lysis reaction substantially improves handling and recovery of expressed proteins by decreasing the viscosity of cell lysates. Product Specifications Storage: Store the Enzyme Mix from the Easy- Lyse Bacterial Protein Extraction Solution at -20 o C. The Lysis Buffer and MgCl 2 Solution may be stored at 4 o C for ease of use. Contaminating Activity Assays: All components of the EasyLyse Bacterial Protein Extraction Solution are free of detectable exo- and endonuclease and RNase activities except for the inherent nucleolytic properties of the Enzyme Mix. Related Products: The following products are also available: PeriPreps Periplasting Kit Ready-Lyse Lysozyme Solution OmniCleave Endonuclease References: 1. Jarvis, B.W. (2003) Epicentre Forum 10 (3), 4. EasyLyse Bacterial Protein Extraction Solution Contents The EasyLyse Bacterial Protein Extraction Solution contains enough reagents to lyse 500, 1 ml bacterial cultures, or the 100 µl cultures in 48 microplates each with 96 wells. EasyLyse Lysis Buffer...50 ml (10 mm Tris-HCl [ph 7.5], 1 mm EDTA, 1% non-denaturing detergent) EasyLyse Enzyme Mix µl 1 M MgCl 2 Solution µl EasyLyse, PeriPreps, Ready-Lyse and OmniCleave are trademarks of EPICENTRE, Madison, Wisconsin. continued Lit. #196 (800) (608) Fax (608)
5 EPICENTRE EasyLyse Bacterial Protein Extraction Solution Protocol for the Lysis of less than 1 ml of Bacterial Culture One milliliter (up to 4 OD 600 or 16 mg cell pellet) of bacterial cell culture may be processed as described below. For proportionally fewer or more bacteria, the reagent amount can be scaled accordingly. For 100 ml of bacterial culture in each well of a 96-well plate, use 20 ml of EasyLyse Bacterial Protein Extraction Solution per well. For 1 ml of bacterial culture in a 1.5 ml microfuge tube, use 200 ml of EasyLyse Bacterial Protein Extraction Solution. 1. Pellet 1 ml of the cells in a microcentrifuge. (1 ml of E. coli cells [at 10 9 cells/ml] will yield approximately mg total cell protein.) 2. Remove the supernatant fluid and freeze the pellet at -20 o C. 3. In a tube add in order: 0.5 ml of distilled water or buffer with protease inhibitors* and 2 ml of 1 M MgCl 2. Mix, and then add 0.5 ml of Lysis Buffer and 1 ml of Enzyme Mix. Mix by gentle pipetting. This mixture is enough for 5 x 1 ml of bacterial culture. 4. Thoroughly resuspend the cell pellet in 200 ml of the above solution. 5. Incubate 5 minutes at room temperature. 6. Pellet the cellular debris by centrifugation in a microfuge for 2 minutes. 7. Transfer the supernatant fluid to a clean tube. Notes: Prepare only as much EasyLyse as needed in the next few hours. Long-term storage may cause the solution to become viscous. * Protease inhibitor cocktails containing EDTA are incompatible with the enzymatic removal of viscosity. Such cocktails may be added after step 5. page 2 2/06
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