2. www.amgenbiotechexperience.com Lisa Yoneda Mira Mesa High School Teacher – Biology to AP Biology – Biotechnology Background: – BS Molecular Biology – LSSI alumni (2 nd year) – Coordinate UCSD ScienceBridge Kits

3. LABORATORY 6: GETTING WHAT WE NEED-PROTEIN LSSI Alum Lisa Yoneda, Biotechnology Program, Mira Mesa HS

4. www.amgenbiotechexperience.com Safety General Lab Safety Guidelines Use laboratory coats, safety glasses and gloves as appropriate Avoid restrictive clothing and open-toed shoes No eating or drinking in the lab Make sure that students are familiar with the operating instructions and safety precautions before they use any of the lab equipment Check all MSDS (Material Safety Data Sheets) for all chemicals and reagents in the lab before preparing and running the lab Wash hands at the conclusion of the lab. Lab Safety Guidelines for lab 6 When using potentially bio-hazardous materials work in a sanitary manner and treat all waste as a potential biohazard Dispose of pipette tips and all other materials that came in contact with bacteria in the biohazard bag provided Any item potentially contaminated by bacteria should be treated with 70% ethanol or another acceptable disinfectant

5. www.amgenbiotechexperience.com Lab Prep & Aliqouting Guidelines Reagents/SuppliesAliquotStorage TempNotes 1 flask of 250 mL of LB/Amp/Ara broth (OR use plate from Lab 5 to inoculate/Kit N/A4oPlease return flask 10 Resin packed columnsN/ARTPlease return columns 10 tubes of 15 mls of Elution Buffer/ kit (EB)N/ART 50 mls extra/kit Please return ALL tubes 10 tubes of 15 mls of Wash Buffer/kit (WB)N/ART 50 mls extra/kit Please return ALL tubes 10 tubes of 15 mls of Column Equilibration Buffer/kit (CEB) N/ART 50 mls extra/kit Please return ALL tubes 10 tubes of 5 mls of Binding Buffer/kit (BB)N/ART 20 mls extra/kit Please return ALL tubes 10 tubes of 15 mls of Ethanol/kit (EtOH)N/ART 50 mls extra/kit Please return ALL tubes 1.6 mls of Lysis Buff/class (LyB)150ul/group4o Equipment/Supplies 10 Student boxes with the following: 1 p20 micropipette 1 microfuge rack 1 p200 micropipette 1 bag of microfuge tubes 1 p1000 micropipette 1 bag of microfuge tubes 1 waste and 1 box of refillable tips (2 ul-200 ul) 1 ice bucket 4 Mini centrifuges 1 Tabletop centrifuge 1 Water bath 10 Ring stands with clamps 10 Boxes of p1000 tips

6. www.amgenbiotechexperience.com Warm Up Why is your transformed bacteria red? – What did you do? – What did the bacteria do?

7. www.amgenbiotechexperience.com Transformation

8. www.amgenbiotechexperience.com Transcription & Translation Where is rfp? Is rfp the only protein made by the cell?

9. www.amgenbiotechexperience.com Why purify a protein? The protein is in living cells and mixed in with other proteins, over 1, 000 proteins could be in one cell Pharmaceutical companies want one purified protein to sell as a medicine. Don’t want other proteins interfering with the medicine or body chemistry

10. Protein Purification ProcedurePurpose 1.Centrifuge 2.Lyse cells- overnight incubation 3.Centrifuge 4.Column Purification increase concentration Release cell proteins (including rfp) Separate large cell pieces from released proteins Separate rfp from other cell proteins

11. www.amgenbiotechexperience.com Where’s your protein? How will you know where your protein is? Each step of the procedure – Record where rfp should be (prelab) – As you complete- check location of rfp!

12. Protein Purification ProcedureWhere is rfp? 1.Centrifuge 2.Lyse cells- overnight incubation 3.Centrifuge 4.Column Purification Cytoplasm inside the cell Liquid portion (cells cut open) Supernatant (liquid) Attach to resin in column and then release

13. PART A (DAY 1) Step 1: Centrifuge Step 2: Lyse cells

14. Outline Protocol (prelab) ProtocolNotes/Changes Be sure to note where the rfp should be for each step Flow chart of each step – Enough details that can use their notes to do the lab

15. www.amgenbiotechexperience.com Part A (Day 1) Overview Step 1: Centrifuge – 1 mL of rfp to microfuge tube – Centrifuge at high speed for 5 min – Remove supernatant without disturbing pellet – Repeat in the SAME microfuge tube!

16. www.amgenbiotechexperience.com Step 1: Centrifuge What will happen when you centrifuge your cell culture?

17. www.amgenbiotechexperience.com Step 1: Centrifuge Flow Chart

18. www.amgenbiotechexperience.com Part A (Day 1) Overview Step 2: Lyse cells – Add 150 µL EB (buffer) to cell pellet – Re-suspend cells No clumps- use spiral notebook Increase surface area for lysozyme – Add 150 µL LB (lysozyme) to cells and mix- use spiral notebook – Incubate Overnight (room temp) Can freeze if longer

19. www.amgenbiotechexperience.com Step 2: Lyse Cells What will happen when we lyse the cells?

20. www.amgenbiotechexperience.com Step 2: Lyse Cells Flow Chart

21. www.amgenbiotechexperience.com Complete Part A (Day 1) Step 1: Centrifuge Step 2: Lyse cells

22. Day 2 Background Understanding Columns

23. www.amgenbiotechexperience.com Warm Up Many medicines today are proteins. Biotechnology companies make these medicines the same way you transformed the bacteria to make rfp. – However, the protein is in the bacteria, so how do we get our protein out without destroying it’s properties? – What effects a proteins ability to do it’s job?

24. www.amgenbiotechexperience.com Understanding purification Methods: Hydrophobicity is often used to help separate molecules. – Hydrophobic: ‘fears water’ : oil, wax, fats – Hydrophilic: ‘loves water’ : salt, sugar Proteins have both hydrophobic and hydrophilic parts – Hydrophilic regions point outward – Hydrophobic regions point inward – (reversed for membrane proteins)

25. www.amgenbiotechexperience.com With over 1,000 different proteins how can we isolate rfp? We used an expression vector The cells are making much more rfp than any other protein Column chromatography: We need to understand the amino acid make up of the rfp Hydrophobic and hydrophilic regions

26. www.amgenbiotechexperience.com RFP Structure What will happen if you place a folded protein in a high salt solution?

27. www.amgenbiotechexperience.com Column Structure Column Stop Cock Matrix Solvents

28. www.amgenbiotechexperience.com Column Structure Column – Glass (or plastic) tube that holds matrix Stop Cock – Regulates flow of solvents In line: allows solvents to flow Perpendicular: blocks solvents- stops flow – Use cap if no stop cock

29. www.amgenbiotechexperience.com Column Structure Matrix – Other names Resin Stationary phase – Coated beads that can bind and release your molecule under different conditions – Note: Must be kept in liquid at all times – Keep 2 mm (~ ½ pinky nail) of liquid above the resin at all times Never disturb resin – Add solvents slowly and down side of the column- no clouds

30. Column Structure SolventsBuffers Equilibration Binding Wash Elution Storage Prepares matrix for binding to molecule of interest Usually mixed with supernatant before adding to matrix- prepares rfp for attaching to resin Removes other molecules from matrix- allows rfp to stay bound Rfp detaches and flows out of matrix Keeps resin stable when not in use

31. www.amgenbiotechexperience.com Binding Buffer High Salt Buffer – Mix with rfp supernatant – Rfp exposes inner hydrophobic amino acids – Rfp and hydrophobic proteins will bind to resin – Hydrophillic proteins will flow through Notes: – Flow through = clear = waste – Ideally add solvent slowly- rfp will bind in tight ring- increasing final concentration

32. www.amgenbiotechexperience.com Wash Buffer Medium Salt Buffer – Allows some refolding of proteins- but not rfp – Moderately hydrophobic proteins release and flow through – RFP will stay bound to resin Notes: Flow through = clear = waste Gravity flow can be slow

33. www.amgenbiotechexperience.com Elution Buffer Rfp will refold- covering hydrophobic amino acids Rfp releases and flows through Note: – Add slowly and all rfp will release and flow through together – Only catch pink/red solution with clean tube – Clear solution = buffer only = waste

34. www.amgenbiotechexperience.com Column Prep Setup columns – Arm/finger clamp – Ring stand – Waste container – Column tip should be just above waste container Drain Storage buffer Add and drain 2 mL of equilibration buffer Note: ALWAYS leave ~2 mm of solution above resin – This is when you add next buffer – Never drain resin dry

35. PART B (DAY 2) Step 3: Centrifuge Step 4: Column

36. Outline Protocol (prelab) ProtocolNotes/Changes Be sure to note where the rfp should be for each step Flow chart of each step – Enough details that can use their notes to do the lab

37. www.amgenbiotechexperience.com Part B (Day 2) Jobs Step 3: Centrifuge (1 group member) Step 4: Column – Column prep (1 group member- 1 st class only) – Run column Step 5: Column Storage/Prep for next class Timing is key! 3 group members needed – Centrifuge, Column prep, gather supplies – Run column (all members together) – Prep/Store column, clean up, check results

38. www.amgenbiotechexperience.com Part B (Day 2) Overview Step 3: Centrifuge (1 group member) – Separate cytoplasm proteins from cell walls and membranes – 200 uL supernatant into clean tube – Add 200 uL binding buffer (BB) – Optional extension: Add 25 uL of bromocresal green Visual demonstration of separation

39. www.amgenbiotechexperience.com Step 3: Centrifuge What will happen when we centrifuge the lysed cells?

40. www.amgenbiotechexperience.com Step 3: Centrifuge Flowchart

41. www.amgenbiotechexperience.com Part B (Day 2) Overview Step 4: Column – Column prep (1 group member- 1 st class only) Drain storage buffer Run Column Equilibration Buffer (CEB) – Run column Binding Buffer (BB) Wash Buffer (WB) Elution Buffer (EB)

42. www.amgenbiotechexperience.com Step 4: Run Column Where is rfp for each step? – Run column Binding Buffer (BB) Wash Buffer (WB) Elution Buffer (EB)

43. www.amgenbiotechexperience.com Always keep ~ 2 mm of solution above resin – then add next solution Only collect red liquid- let clear flow into waste Add buffers slowly down sides- no clouds Step 4: Run Column Flowchart

44. www.amgenbiotechexperience.com Optional Extension Do rfp and bromocresal green come out of the column at the same time? – Why or why not? – Which one is more hydrophobic? How do you know?

45. www.amgenbiotechexperience.com Step 5: Column Storage Storage – Run 2 mL of Storage Buffer (20% ETOH) – Add 1 mL of storage buffer to top of resin – Replace top & bottom caps Top cap should snap on tightly Another class – Run 2 mL of column equilibration buffer (CEB) – Replace top & bottom caps Top cap should snap on tightly

46. www.amgenbiotechexperience.com Part A (Day 1) Teacher Prep Notes Can pre aliquot 1000 ul rfp into student tubes (1/group) – Have students pre-label tubes – Some down time- can have previous class aliquot next class solution – 1 st to class gets to aliquot Pre-label collection rack (1/period) If have sensitive centrifuge recommend having students use a balance for 2 nd spin

47. www.amgenbiotechexperience.com Part B (Day 2) Teacher Prep Notes Columns run slow – Can prep columns ahead of time Students Centrifuge & setup column at same time Slow columns – Make sure stop cock open Caps (top & bottom) are removed – Unclog by resettling resin – Apply pressure

48. www.amgenbiotechexperience.com Applying Pressure Can make plunger to increase column flow rate – #2 stopper- 1 hole – 10 mL syringe – Attach together by cutting 1 mL disposable pipet Using plunger – Never pull up on plunger while attached to column – Needs tight seal- columns can be warped into oval shape Check if clamp is too tight Gently squeeze column into round shape – Make sure you still have drops, not a steady stream coming from column when using

49. www.amgenbiotechexperience.com Video Links for Lab 6 Lab 6 Purification Help – Lysing the cells (quantities differ) Lysing the cells – Column Protein Purification Overview Column Protein Purification Overview – Eluting rfp Eluting rfp Column Help – Unclogging columns Unclogging columns – Column basic setup & mistakes Column basic setup & mistakes