THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY. STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 110 MOD: 2 nd Issue Page: 1 of 17

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1 THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 110 MOD: 2 nd Issue Page: 1 of 17 Procedure Type: General Laboratory Procedure 1. Risk Assessment: This Risk Assessment is to be used as a general guide and as such, cannot accommodate all the varying factors that may be encountered when using this procedure. Therefore, personnel are requested to conduct their own Risk Assessment before using this procedure to include any extra hazards introduced by the task performed. TASK PERFORMED Two dimensional separation of proteins - 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) is a method that labels protein samples with fluorescent dyes before 2-D electrophoresis, enabling accurate analysis of differences in protein abundance between samples. HAZARDS 1. Chemical hazard acrylamide Toxic 2. Chemical hazard bis-acrylamide Toxic, harmful 3. Chemical hazard methanol Toxic, flammable 4. Chemical hazard acetic acid Corrosive, flammable 5. Chemical hazard sodium dodecyl sulphate Irritant 6. Chemical hazard thiourea Danger to the Environment, Toxic 7. Chemical hazard TEMED Highly Flammable, Corrosive 8. Chemical hazard APS Oxidising agent, Harmful 9. Chemical hazard TBP Irritant 10. Chemical hazard 2D Quant kit, 2D Cleanup kit precipitant Corrosive, Danger to the Environment 11. Chemical hazard 2D Quant kit - copper solution Irritant, Danger to the Environment 12. Chemical hazard 2D Cleanup kit wash buffer Flammable, Carcinogenic, Harmful, Irritant 13. Chemical hazard sodium hydroxide Corrosive NAME (signed) WRITTEN BY Nikki Verrills REVIEWED BY.. Lynn Herd.. Phil Dickson DATE 16 th March th June th October 2008 Distributed To: GLP Master File / GLP Lab File

2 Page: 2 of Chemical hazard CyDyes Cy2, Cy3, Cy5, Harmful 15. Chemical hazard dimethylformamide Flammable 16. Chemical hazard IPG Strip Holder Cleaning Solution Harmful, irritant 17. Chemical hazard 70% ethanol Flammable 18. Chemical hazard butanol Flammable, harmful 19. Chemical hazard iodoacetamide Toxic, harmful 20. Electrical hazard electrophoresis / isoelectricfocusing equipment 21. Biological hazard samples 22. Burns molten agarose RISK ASSESSMENT 1. The risk of chemical exposure is reduced by consulting the risk assessments for each of the individual chemicals identified. Follow controls outlined in risk assessment. 2. The risk from electrical equipment is low if the risk controls are followed. 3. The risk from biological samples is moderately low if the risk controls are followed. 4. The risk from burns from molten agarose is low if the risk controls are followed. RISK CONTROL 1. Refer to Chemical Risk Assessments for each of the chemicals identified as hazardous. 2. Minimum PPE required is lab coat, enclosed shoes, disposable gloves and safety glasses. Further PPE may be identified in individual risk assessments. 3. Acrylamide and Bisacrylamide in their monomeric forms are neurotoxic and all contact with these chemicals should be minimised. Use in a fume cupboard where possible and wear nitrile disposable gloves. When dealing with the powdered form use a particle mask. 4. Methanol, ethanol, butanol and TEMED are flammable chemicals. Keep away from spark and open flame and use minimal quantities only. 5. Acetic acid is corrosive. Minimise quantities. 6. Prepare 7% acetic acid / methanol Gel Fixing / Destaining solution in fume cupboard. This solution can be used in a lidded container in the workarea whilst fixing or destaining. 7. SDS is a very light surfactant. Ensure that particle mask is worn when weighing out dry chemical. 8. Thiourea is used in DIGE Lysis buffer and sample rehydration buffer. It is a possible carcinogen and reproductive toxin. Minimise all exposure to the stock chemical and to the resulting solutions. PPE including safety glasses and disposable gloves must be worn. Wherever possible work with this chemical in a fume cupboard. Weighing of crystals should take place in the SafeWeigh balance enclosure.

3 Page: 3 of APS is an oxidizing agent. Keep away from combustible materials. 10. TBP tributylphosphine is an irritant and should be used in the fume cupboard. Do not store residual working solution following procedure, but dispose immediately into labeled waste container. Final disposal via University chemical waste collection. 11. Dimethylformamide is a reproductive/developmental toxin, Minimise all contact with this chemical. Use in a fume cupboard is recommended. 12. IPG strip holder cleaning solution is an irritant. Wash thoroughly after handling. 13. Sodium hydroxide solution and glacial acetic acid are corrosive. Minimise handling and prepare concentrated solutions in fume cupboard D Quant Cleanup kit precipitant corrosive, dangerous to the environment wear safety glasses and disposable gloves. Collect solution for disposal by licensed chemical waste contactor D Quant kit copper solution dangerous to the environment collect solution for disposal by licensed chemical waste contactor D Cleanup kit wash solution is highly flammable and should be used in fume cupboard. 17. CyDyes wear particle mask and disposable gloves when handling. 18. Iodoacetamide limited evidence of toxicity. Use particle mask when weighing out powder. 19. Check all electrical leads for damage. 20. Ensure electrical goods are handled with care and electrophoresis apparatus is set up PRIOR to power pack being switched on. 21. The power supply must be shut off prior to removing the lid, handling leads or disconnecting plugs from the apparatus, and any voltage allowed to dissipate. 22. Always run the electrophoresis unit with the lid in place over the buffer chamber and gel. Do not attempt to use the unit without the safety lid. 23. Treat all human samples as potentially infectious. Wear lab coat, safety glasses and latex gloves at all times when handling samples. 24. Avoid production of aerosols from human samples. 25. If possible work in a biological safety cabinet. 26. Personnel should be advised to be up to date with Hepatitis B and Tetanus vaccinations. 27. Agarose should be heated in microwave until redissolved and then handled using leather heat-protective gloves. Ensure agarose is stored in Schott bottle suitable for reheating 28. Training should be provided by personnel experienced in this procedure. 29. Training should be undertaken in General Laboratory Safety.

4 Page: 4 of Purpose: 2.1. Two-dimensional separation of proteins. One dimension iso-electrofocusing, the second dimension electrophoresis. 3. Equipment: 3.1. Sonicator 3.2. Centrifuge 3.3. Rehydration tray 3.4. IPGphor IEF system (Ettan) 3.5. vortex 3.6. cleaning brush 3.7. vortex 3.8. low fluorescent glass plates 3.9. Dalt-12 caster cm petri dish shaking platform DALT 6 tank Power pack Wonder bar glass plate separator Typhoon scanner large tupperware container 4. Materials: 4.1. Cells in T75 tissue culture flask 4.2. Esky of ice 4.3. microfuge tubes 4.4. Dry strips IPGphor 4.5. Electrophoresis wicks 4.6. kimwipes 4.7. gladwrap 4.8. filter paper

5 Page: 5 of Reagents: 5.1. DIGE Lysis Buffer: Reagent MW Quantity Final Conc Tris g 30 mm Thiourea g 2 M Urea g 7 M CHAPS g 4% (w/v) MQ-H 2 O to 50 ml ***ph to 8.5, aliquot and store at -20 o C for up to 3 months PBS mM NaOH 5.4. CyDye x Sample/Rehydration Buffer STOCK: Reagent MW Quantity Final Conc Urea g 7 M Thiourea g 2 M CHAPS g 4% (w/v) MQ-H 2 O to 25 ml ***Aliquot and store at -20 o C, up to 6 months x Rehydration Buffer: 2 x Sample/Rehydration Buffer 2 ml Stock Ampholyte, ph 3-10 (40% stock) 50 µl 1% (v/v) DTT 4 mg 0.2% (w/v) ***Make fresh before use 5.7. Mineral oil 5.8. CyDye DIGE Fluor minimal dye (Cy2, Cy3, Cy5), mM Lysine Reagent MW Quantity Final Conc L-lysine mg 10 mm MQ-H 2 O to 10ml ***Aliquot and store at -20 o C, up to 3 months

6 Page: 6 of x Sample Buffer: 2 x Sample/Rehydration Buffer 1 ml Stock Ampholyte, ph 3-10 (40% stock) 50 µl 2% (v/v) DTT 20 mg 2% (w/v) ***Make fresh before use saturated butanol Gel Solutions: M Tris-Cl, ph 8.8: Reagent MW Quantity Final Conc Tris g 1.5 M MQ-H 2 O*** to 500 ml ***Add MQ-H 2 O to ~400 ml first then ph to 8.8 with HCl. Store at 4 o C, up to 2 weeks % Ammonium Persulphate (APS): APS 1 g 10% MQ-H 2 O*** to 10 ml % Sodium Dodecyl Sulphate (SDS): SDS 10 g 10% MQ-H 2 O*** to 100 ml Displacing Solution: 1.5 M Tris-Cl, ph ml M Glycerol (87% stock) 115 ml 50% Bromophenol Blue Trace MQ-H 2 O 35 ml 200 ml

7 Page: 7 of % SDS-PAGE Gel: 40% Acrylamide/Bisacrylamide 280 ml 12.5% Solution (BioRad), 37.5:1 1.5 M Tris-Cl, ph ml M 10 % SDS 9 ml 0.1 % 10% APS** 4.5 ml 0.05% TEMED 300 µl 0.03% MQ-H 2 O ml 900 ml **Make APS fresh before use, add APS last prior to pouring Add SDS-PAGE solution together, except TEMED and APS, mix thoroughly with a magnetic stirrer. Degas solution, 30 min. Add TEMED and APS into the solution and mix again with magnetic stirrer, sec Saturated Butanol: Reagent Quantity Butan-1-ol 100 ml MQ-H 2 O 100 ml ***Mix butanol and H 2 O in bottle and shake, allow the solution to settle overnight or until two layers are formed. Saturated butanol is the top layer. Equilibration solutions M Tris-Cl, ph 8.0: Reagent MW Quantity Final Conc Tris g 1 M MQ-H 2 O*** to 500 ml ***Add MQ-H 2 O to ~400 ml first then ph to 8.0 with HCl. Store at 4 o C, up to 2 weeks SDS Equilibration Buffer Stock A: Reagent MW Quantity Final Conc Tris-Cl (1.0 M, ph 8.0) 50 ml 100 mm Urea g 6 M Glycerol (87% ml 30% (v/v) stock) SDS g 2% (w/v) MQ-H 2 O to 500 ml ***Store at room temperature, for up to 6 months.

8 Page: 8 of SDS Equilibration Buffer Stock B: Reagent MW Quantity Final Conc Tris-Cl (1.5 M, ph 8.8) 25 ml 375 mm Urea g 6 M Glycerol (100% 20 ml 20% (v/v) stock) SDS g 2% (w/v) Acrylamide (40%) 6.25 ml 2.5% MQ-H 2 O to 100 ml *Aliquot and store at -20deg Equilibration Solution 1: SDS Equilibration Buffer 10 ml* - Stock Solution DTT 50 mg 0.5% (w/v) *10 ml is used per strip Equilibration Solution 2: SDS Equilibration Buffer 10 ml* - Stock Solution Iodoacetamide 450 mg 4.5% (w/v) *10 ml is used per strip Equilibration Solution 3: SDS Equilibration Buffer 10 ml* - Stock Solution B 200mM TBP 250ul 5mM *10 ml is used per strip.

9 Page: 9 of 17 Second Dimension Electrophoresis Solutions: x SDS-PAGE Running Buffer Stock: Reagent MW Quantity Final Conc Tris g 250 mm Glycine M SDS g 0.2% (w/v) MQ-H 2 O to 2L x SDS-PAGE Running Buffer: 10 x SDS-PAGE Running 600 ml* 1 x Buffer Stock MQ-H 2 O 5.4 L 6 L *Volume required for DALT6 is 5.5 L % (w/v) Agarose Overlay Solution: 1 x SDS-PAGE Running 100 ml Buffer Agarose 0.2 g 0.2% Bromophenol blue Few grains Trace % (w/v) Agarose Overlay Solution: 1 x SDS-PAGE Running 100 ml Buffer Agarose 0.5 g 0.5% (w/v) Bromophenol blue Few grains Trace % (w/v) Agarose Sealing Solution: 1 x SDS-PAGE Running 200 ml Buffer Agarose 4 g 2% (w/v)

10 Page: 10 of Gel Fixing/Destaining Solution: Methanol 100 ml 10% (v/v) Acetic Acid 70 ml 7% (v/v) MQ-H 2 O 830 ml 1 L Sypro Ruby stain MW marker 6. Set Up: 6.1. Ensure that you have read and understood the Safety Precautions (Section 6 of this SOP) prior to commencing this procedure. 7. Safety Precautions: 7.1. Good laboratory techniques are to be used at all times Wear appropriate PPE lab gown, safety glasses, disposable gloves and enclosed shoes. 8. Method: 8.1. Sample Preparation cell culture samples Wash cells 3x with PBS (2 x 10 7 cells, ~1 x T75 flask). Proceed immediately to next step or cell pellet can be snap frozen and stored at -80 o C until needed Resuspend cells in DIGE Lysis Buffer (~5-10 mg/ml, 500 µl), incubate lysate on ice for 10 min Sonicate using probe sonicator to completely lyse cells, 10 pulses on setting Centrifuge cell lysate (12,000g, 10 min, 4 o C) and transfer supernatant to new tube Determine protein concentration using 2D Quant Kit (GE Healthcare), assay using 5 µl sample in duplicate Eliminate high salt contents of the samples by using 2D Clean-Up Kit (GE Healthcare). Clean up µg of samples (100 µg/eppendorf tube) Resuspend cleaned samples in DIGE Lysis Buffer at ~5 µg/µl Check cell lysate ph by spotting 0.2 µl onto a ph indicator strip. Adjust to ph ~8.5 with 50 mm NaOH Determine protein concentration using 2D Quant Kit. Samples can be stored at -80 o C until needed.

11 Page: 11 of For the internal standard, pool 25 µg of each protein sample to be analysed, and take 50 µg of each protein sample in separate tubes, ready to be labelled with CyDye Immobiline DryStrip Rehydration In a rehydration tray, pipette ~200 µl (for 11 cm strips) or ~450 µl (for 24 cm strips) of 2x rehydration buffer and place DryStrips gel side facing down onto the buffer Cover strips with ~0.5 ml (for 11 cm strips) or ~1 ml (for 24 cm strips) of mineral oil to prevent evaporation Leave at room temperature, overnight (up to 24 hrs) Preparation of CyeDye DIGE Fluor Minimal Dye and Labelling of Protein Prior to reconstituting CyDye DIGE Fluor minimal dye (Cy2, Cy3, Cy5), bring the lyophilised dyes to room temperature by leaving the tubes on the bench (capped), ~10-30 mins Reconstitute CyDye DIGE in high quality anhydrous dimethylformamide (MF) at 1 nmol/µl Vortex vigorously for 30 sec to dissolve dye and spin for 12,000g, 30 sec Prepare working dye solution at 400 pmol/µl for your samples (400 pmol/50 µg protein sample). Working dye solution is stable at -20 o C, up to 2 weeks Add 1 µl working dye solution to 50 µg of the protein sample Vortex vigorously to mix the protein with the dye, then centrifuge briefly, and incubate in the dark on ice for 30 min. The internal standard is labelled with Cy2 dye Add 1 µl of 10 mm lysine to stop labelling reaction, mix and spin briefly. Incubate in the dark on ice for 10 min Labelled samples can be stored in the dark at -80 o C, up to 3 months Combine the three differentially labelled samples (Cy2, Cy3, Cy5) according to experimental design, mix and spin briefly Add equal (1:1) volume of 2x sample buffer to pooled samples, incubate on ice for at least 10 min First Dimension Isoelectric focusing Place ceramic manifold on the IPGphor IEF system (Ettan) and pour mineral oil in every well until level with each well, do not overfill Place strips in position according to Figure 1, making sure that for 24 cm strips, that the positive end is pushed up to the end where the salt sumps are, which draws out excess salt from the sample.

12 DISCIPLINE OF MEDICAL BIOCHEMISTRY PROCEDURE NO: Page: GLP of 17 Figure 1. First Dimension Isolectric Focussing - Positioning of Strips in IPGphor Manifold Wet 2 wicks per strip by squirting MQ-H2O and place each on the positive and negative ends of the strips. For prep samples where protein load is ~600 µg, place 2 wicks per end to absorb excess salt, or allow focussing to run for a few hours then change to fresh wicks as required Place sample cup at the negative end and make sure there are no leakage by pipetting some mineral oil into the cup Place positive and negative electrodes in place Run 11 cm strips on Program 8 11 cm Sela :Step-n-Hold, 100 ma/strip: 1) 100 V, 3 hr 2) 300 V, 2 hr 3) 600 V, 1 hr 4) 1000 V, 1 hr 5) 2000 V, 1 hr 6) *3500 V, 16 hr 7) 100 V, 5 hr **Strips can be taken off in step 6. Run 24 cm strips on Program 10 Tracy Program#2 24 cm :100 ma/strip: 1) Step-n-hold 300 V, 3 hr 2) Gradient 1000 V, **6-10 hr

13 Page: 13 of 17 3) Gradient 8000 V, 3 hr 4) Step-n-hold 8000 V, 4.4 hr **Step 2 may be adjusted to run for 6 hr or up to 10 hr to accommodate for the time on the next day when strips can be taken off After first dimension run, make sure used mineral oil is poured into the waste flask Ceramic manifold and electrodes must be soaked in warm water, with several drops of the IPGphor cleaning solution, for at least 2 hrs. This is to remove any remnant proteins After soaking, use brush to clean the manifold and electrodes, dry and place back into storage Pouring Large Format SDS-PAGE Gels for 24 cm Strips Pour gels at least a day before use. It is important that the gels are absolutely level at the top in order for good contact to be achieved between the strip and the SDS-PAGE gels Clean low fluorescent glass plates carefully and thoroughly with MQ-H 2 O then 70% EtOH and wipe with Kimwipes Align each glass plate pair such that all sides are flushed, place glass plates into Dalt-12 caster (up to 14 gels can be cast) Make sure caster placed together tight Using funnel, fill caster tank with 12.5% SDS-PAGE solution to ~1inch below desired level Add Displacing Solution until SDS-PAGE solution is at desired level Overlay gel with Saturated Butanol, ~ 4 ml per gel Cover with glad wrap and leave to set overnight Once set, wash away the butanol with H 2 O. Gels can be stored at 4 o C with Running Buffer for up to 2 weeks, or used immediately Rinse Dalt-12 caster with water and dry before putting away Equilibration of Strips For 24 cm strips, place focused strip in a 10 cm petri dish with gel side facing towards the inside of the dish and plastic backing against petri dish side as in Figure 2.

14 Page: 14 of 17 Figure 2. Equilibration of 24 cm strips Equilibrate with 10 ml Equilibration Solution 1, 20 min on shaking platform Discard solution and replace with 10 ml Equilibration Solution 2, 20 min on shaking platform. OR Equilibrate for 20min in Equilibration solution 3, 20min on shaking platform Second Dimension - Electrophoresis Fill DALT6 Tank with 1 x Running Buffer to the first line indicated on the side of the tank Turn on the DALT6 Tank to allow circulation Turn on cooler and set temperature to 10 o C (if running through the day) or 4 o C (if running overnight) Dip equilibrated strip into 1 x Running Buffer Slot strip into poured gel well (see Figure 3).

15 Page: 15 of 17 Figure 3. Second Dimension SDS-PAGE - Positioning of Immobiline Strips in DALT6 SDS-PAGE Gels Pipette 1 ml molten 0.2% agarose overlay solution into the well Tap strip into place such that there are no bubbles between the strip and the gel edge Pipette 1 ml molten 0.5% agarose overlay solution over the strip, leave and let set On a paper wick or filter paper pipette the MW standard and let dry completely Insert paper into the gel well, at the end of strip Using a squirt bottle, squirt both sides of the gel glass with 1 x Running Buffer and insert into the slot of the upper chamber in the DALT6 tank. If you are running less than 6 gels, fill up the remaining slots with the blank cassettes Seal the upper chamber with 2% agarose gel Fill the upper chamber with 1 x Running Buffer such that the bottom chamber and upper chamber are level with each other, i.e. just past the maximum fill level indicated on the side of the tank Place the DALT6 lid on and connect the electrodes to the powerpack Run gels with the following conditions:- For running within the day, Max Voltage, Max Current

16 Page: 16 of 17 1) Step W/gel, 1 hr 2) Step W/gel, 1 hr 3) Step 3 17 W/gel, 8-10 hr For running overnight, Max Voltage, Max Current 1) Step W/gel, 2 hr 2) Step 2 1 W/gel, 16 hr When gel front is ~1 cm from the bottom, it can be stored at 4 o C overnight to 24 hrs or scanned immediately Pour away 1 x Running Buffer from DALT6 tank and rinse everything with water then allow to dry before storing Scanning Gels Wipe clean both sides of the glass plates of the 2D-DIGE gels with MQ-H 2 O then with 70% EtOH using Kimwipes. It is important that no dust is left on the glass as these may autofluoresce and appear as false protein spots Place glass plates carefully onto the scanner bed, using the gel guides. Two gels can be scanned at once Gels are scanned on the Typhoon Scanner under the following fluorescence settings: i. Cy2 520 BP 40 Blue2 (488) ii. Cy3 580 BP 30 Green (532) iii. Cy5 670 BP 30 Red (633) Check Press Sample, Focal Plane of +3mm Select Tray DIGE Ettan DALT, this scans 2 plates Use the DIGE naming system Do a pre-scan at 1000 micron pixels to check for the maximum balance of the CyDyes Adjust the PMT of each CyDye such that there are no saturated protein spots and all dyes are at about the same intensity, usually PMT Once balance is achieved, scan gels at 200 micron pixels (this takes ~30 min) Staining Gels Carefully remove gels from the glass plates using the wonder bar glass plate separator.

17 Page: 17 of Place into a large tupperware container Fix gel with Fixing Solution (enough to just cover gel), incubate at room temperature on a shaking platform, 30 min Discard Fixing Solution and stain gel with Sypro Ruby (enough to just cover gel), incubate in the dark (covered with foil) at room temperature on a shaking platform, hrs Take Sypro Ruby aside (Sypro Ruby can be re-used up to five times) and destain with Destaining Solution, room temperature on a shaking platform, 30 min Wash gel with water, room temperature, on a shaking platform until ready to scan Scanning Sypro Ruby Stained Gels Place gel onto scanner bed, careful not to introduce any air bubbles Scan at 200 micron pixels, under flu 9. Maintenance: 9.1. Clean all equipment used and return to storage location 10. Shutdown: Dispose of chemicals according to the disposal instructions in the Risk assessments Dispose of polyacrylamide gels into contaminated waste bins. 11. Review Date: This procedure should be reviewed before 24 th July 2011, or on the introduction of any change to the method. 12. Change History: Issue Number: 1st Issue Date Issued: 23 rd March Issue Number: 2 nd Issue Date Issued: 19 th November 2008 Reason for Change: Revision of risk assessment