soptfmsdeglycosylationdoc.doc

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1、SAFE OPERATING PROCEDURETrifluoromethanesulfonic acid (TFMS) Deglycosylation (based on protocol from PROzyme/Glyko Glycofree Chemical deglycosylation kit GKK-500)Any accidents must be reported immediately to Carolyn Schultz 37342 (office) 37392 (lab) or Gary Taylor ext 37275General TFMS deglycosylat

2、ion requires use of various hazardous chemicals. You should be familiar with the MSDS of all chemicals to be used before starting procedure. This SOP only details TFMS, toluene and pyridine as they are not commonly used. The method also includes the use methanol, acetone, ammonium bicarbonate, dry i

3、ce and ethanol.TFMS is one of the strongest acids known. It is a colourless to faint yellow liquid.Toluene is a colourless liquid, flashpoint 4C, autoignition 535C, boiling point 110C, melting point -93CPyridine is a colourless to very faint yellow liquid. Flashpoint 17C, autoignition 482C, boiling

4、point 115C, Melting point -42CHazardsTFMS: Class 8 corrosiveCauses burns, risk of serious damage to eyesToluene: Class 3 flammableHarmful if swallowed, irritating to eyes and skin. Vapour may cause dizziness or suffocation or drowsiness. May produce discomfort to respiratory system. Inhalation or sk

5、in contact may produce health damage. Possible risk to unborn child, danger of serious damage to health by prolonged exposure through inhalation.Pyridine: Class 3 flammableIrritating to eyes, harmful by inhalation, in contact with skin and if swallowed. Can be absorbed through skin, may produce disc

6、omfort of respiratory system and skin. Possible skin sensitiser, limited evidence of carcinogenic effect.Risk Control MeasuresTFMS/toluene/pyridine:Consult MSDS prior to useWear suitable protective clothing (eg nitrile gloves, safety glasses, lab coat)Only use in fume hoodAlert others to use of thes

7、e chemicals with sign indicating the hazardEngineering / Ventilation ControlsLocal exhaust ventilation (eg fume hood) recommendedStorage RequirementsTFMS: Keep locked up, container tightly closed.Toluene: Keep container tightly closed away from sources of ignitionPyridine: Keep container tightly clo

8、sed away from sources of ignitionFirst Aid / Spill Control ProceduresEye: Wash with running water for 15 minutes. Seek medical attentionSkin: TFMS/pyridine: Flood body with water. Remove contaminated clothing. Wash with water and soap. Seek Medical Attention Toluene: Remove contaminated clothing. Wa

9、sh with water and soapInhaled: Fresh air. Rest, keep warm. If breathing shallow, seek medical attention.Poison Information Centre phone 131126Wear protective clothing.Small spill: Do not attempt clean up if you feel unsure of your ability to do so or if you perceive the risk to be greater than norma

10、l laboratory operations.Prevent from entering drains. Eliminate ignition sources, absorb with dry agent (TFMS neutralise with soda ash/lime). Contain spillage by any means. To clean floor/objects, use water (TFMS & toluene- also use detergent)Large Spill: Notify others in the area of spill. Evacuate

11、 area. Notify Dept Safety Officer (Gary Taylor 37275), Carolyn Schultz 37342 (office) 37392 (lab) and Campus Security (37200). Barricade area with tape (in Spill Kits) to prevent entry until arrival of response personnel. Provide assistance and information to spill clean up crew.WasteAll waste to be

12、 collected following the University of Adelaide Waste Disposal procedure. Collect liquid and solid waste separately. Contact Carolyn Schultz 37342 (office) 37392 (lab) for current waste disposal instructions.Applicable Standards and RegulationsA computer based chemical management system (Chemwatch)

13、is available through the University webpages. The address is http:/chemwatch.adelaide.edu.au.National Code of Practice for the Control of Workplace Hazardous Substances NOHSC:2007 (1994.Occupational Health and Safety (Hazardous Substances) Regulations 1999Australian Standard AS 2243.10 (Safety in la

14、boratories - Storage of chemicals).Trifluoromethanesulfonic acid (TFMS) Deglycosylation (based on protocol from PROzyme/Glyko Glycofree Chemical deglycosylation kit GKK-500)*Read SOP, all relevant MSDS and entire procedure before starting*Need:TFMS1Sigma-Aldrich cat. # 347817 $69/5gToluene2Sigma-Ald

15、rich cat. # 244511$43.50/100mLPyridineSigma-Aldrich cat. # P57506$90/1LAmmonium BicarbonateCrown Scientific cat. # 897-500G$36MethanolAcetoneDry Ice (and ethanol for bath)Caltex service station (Hutt St)$9.90/5kgOr can buy 24+kg from Air Liquide at $2.50/kg Nitrile gloves (powder-free, chemically re

16、sistant)Reaction vials (12x33mm), caps and septa (Teflon coated) available from variousSyringe (glass, gas-tight with Teflon plunger and stainless steel needle)All chemicals should be reagent grade. TFMS & toluene must be anhydrous and only dispensed using a chemically inert syringe. All chemicals o

17、nly to be opened in fume hood and any waste to be disposed of via Collex.1As you will only use a small amount of TFMS, look into buying smaller ampules (may take longer to come in). 2To keep toluene dry after opening, add molecular sieves (Im using Type 5A 4-8 mesh beads, but any types smaller would

18、 also be suitable)Prior to deglycosylationEnsure samples are completely dry in reaction vials (sealed with cap and septa) and are relatively free of salts, metals and detergents. Sample quantity should be in range of 0.25-1mg. If any water present, you may get acid hydrolysis of the peptide backbone

19、.Dry syringe thoroughly by washing with dry, reagent grade acetone, flush with dry nitrogen, warm in a dry oven (90C), cool and store in desiccator.Prepare an ethanol/dry ice bath. Add pieces of dry ice to a suitable dish (ie Styrofoam box), slowly add ethanol till it just covers ice. Attach a sign

20、to warn others of hazard.Reagents: Open/prepare all reagents immediately prior to use, TFMS can only be open a maximum of 5 minutes before use (use fresh TFMS each time). Use in Fume HoodDeglycosylation(All steps to be done in a Fume Hood)1. Open TFMS and add toluene to final concentration of 10% (t

21、ransfer 180mL TFMS to vial and add 20mL toluene) (aliquot remaining TFMS into glass vials after finish adding mix to samples!)2. Gently mix, and rinse syringe once with mixture3. Place samples in ethanol/dry ice bath (so they rest firmly on dry ice at side of vessel) and allow to cool for 20 seconds

22、4. Use syringe to add 50mL TFMS/toluene mix to each sample by letting it run down side of vial over 15-20 seconds (some may freeze on side).5. Place vials at -20C (in a container, labelled with hazard)6. After 5 minutes, shake briefly to aid melting of contents and solvation of sample7. Shake after

23、another 5 minutes8. Leave vial in freezer for 4 hoursNeutralisation of Excess TFMS(All steps to be done in a Fume Hood)Prepare pyridine solution: pyridine:methanol:water at ratio of 3:1:1 (e.g. For 600ml mix: 360mL pyridine + 120mL methanol + 120mL water) 1. Remove samples from freezer and remove ca

24、ps and septa (neutralisation is highly exothermic so this allows gas to escape)2. Place vials in ethanol/dry ice bath and leave for 20 seconds3. Using pipette, add 150mL of pyridine solution by letting it flow down side of vial over 15-20 seconds. Leave in bath for at least another 20 seconds4. Tran

25、sfer vial to dry ice for 5 minutes5. Transfer to wet ice for 15 minutes6. Transfer sample to 2mL eppendorf tube7. Add 400mL of neutralisation solution (if solution left in glass vial, rinse and transfer to 2mL tube) and mix briefly (0.5% ammonium bicarbonate)Here the polypeptide may form a precipita

26、teRecovery of deglycosylated polypeptidePrecipitationUse this procedure if precipitate formed during neutralisation.1. Cool sample to 4C and incubate for 30 minutes2. Centrifuge at max speed for 15 mins at 4C3. Keep supernatant in case not all has precipitated4. Resuspend pellet in cold buffer(?) and spin5. Repeat at least 3 more times.Keep all supernatants, pool, evaporate and reconstitute with buffer. Isolate remaining peptide using dialysis or gel filtrationAlternative methods: Gel Filtration (e.g. PD-10) or Dialysis29c295fcc301f42b2b3e156e1b466a9f.doc.Created on 29 十一月 2006 . Page 4 of 4