Laboratory - Purification of Beta Galactosidase | Chem 472, Lab Reports of Chemistry

Download Laboratory - Purification of Beta Galactosidase | Chem 472 and more Lab Reports Chemistry in PDF only on Docsity! Purification of Beta-galactosidase Materials Cell pellets (in freezer) HisTag Binding buffer (20mM TRIS/HCl, 0.5M NaCl, 5mM Imidazole, pH 7.9) HisTag Wash buffer (20mM TRIS/HCl, 0.5M NaCl, 40mM Imidazole, pH 7.9) HisTag Elution buffer (10mM TRIS/HCl, 250mM NaCl, 0.5M Imidazole, pH 7.9) Centrifuge Sonicator Column apparatus Iminodiacetic acid NiCl2 Gel electrophoretic apparatus SDS Solution Incubator Gels (Prepare ahead of time.) ONPG solution (4mg/mL) Z-buffer (60mM sodium phosphate, pH 7.0; 10mM KCL: 1mM MgSO4: 50mM bME) 1 M sodium bicarbonate Bradford Reagent (100mg Coomassie Brilliant Blue G-250, 50mL 95% ethanol, 100mL 85% phosphoric acid) Standard: Chicken Egg Ovalbumin (or BSA) I. Procedure Preparation 1. Make sure all buffers are made. 2. Make sure you have a gel prepared (Skip this step if column is already prepared) Column Preparation 1. Pour 1mL of Iminodiacetic acid into column, yielding 1 column volume of solid (after run through), which is equal to approximately 0.5 mL. 2. Add 1mL of NiCl2 to column and allow to run through. 3. Wash with HisTag binding buffer for a total of 6 column volumes, (3 washings of 2 column volumes each). (1 column volume is equal to 0.5ml) 4. Clamp column, leaving wet. If column is left overnight, add 1-2mL of 20% ethanol to column and clamp. This keeps the column from drying out with time and also inhibits bacterial growth. Sonication – Cell Lysis 1. Add 2mL of HisTag binding buffer to frozen pellet(s) in a tall 15 mL conical and mix by vortexing. 2. Locate sonicator under the fume hood. 3. Use wrench to unscrew cap. Place cap to side. (Replace when finished with sonication.) 4. Screw in lead tip. 5. Turn power switch on (located on bottom right of machine). 6. Wait and let system run its checkpoints. 7. Press “select” button that is second from the top. 8. Cursor should be flashing in the time area. 9. Enter: 3, 0, 0. Hit enter. It should look like: 00:03:00. 10. Press the “” button. 11. Press the first select button at the top. 12. Enter: 1,0. Hit enter. It should look like: 00:00:10. 13. Press the second “select” button from the top. 14. Enter: 5,0. Hit enter. It should look like 00:00:50. 15. Press the “” button. 16. Te program is now set. Don’t touch any other buttons on the machine. 17. Fill a large bucket with ice. Place the conical with the buffer/pellet mixture into the ice and leave it throughout sonication. 18. Place the sonication tip as close to the bottom of the liquid without touching the bottom of the conical itself. If during sonication the mixture becomes bubbly or whipped (looking similar to meringue), stop sonicator and readjust the tube to where the sonicator tip is positioned properly. 19. Press the start button. Wait for sonication to stop before you take the sample from the tip. Do not touch the tip! 20. Turn the machine of. 21. Clean and disassemble the tip and replace the cap. 22. Pipette the liquid from the conical into microcentrifuge tubes. 23. Evenly distribute the microcentrifuge tubes into the centrifuge. Centrifuge at a high speed at 14,000 rpm for 5 minutes. 24. Remove supernatant from tubes and save in new tubes labeled “lysate.” Place tubes into a tube rack and refrigerate until ready to proceed to the next part of the experiment. 25. Take one “lysate” tube of supernatant and save for assays & electrophoresis later. Purification – Column 1. Uncap and load lysate sample and collect effluent in tube 1. 2. Re-clamp column and pour in 1mL of wash buffer unclamp and collect effluent in tube 2. 3. Re-clamp column and pour in 1mL of wash buffer unclamp and collect effluent in tube 3. 4. Re-clamp column and pour in 1mL of wash buffer unclamp and collect effluent in tube 4. 5. Re-clamp column and pour in 1mL of elution buffer unclamp and collect effluent in tube 5.