THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY. STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 072 MOD: 1st Issue Page: 1 of 6

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1 Page: 1 of 6 Making Polyacrylamide gels - Ashman 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 Preparation of SDS PAGE gels for electrophoresis. HAZARDS 1. Chemical hazard acrylamide Toxic / Carcinogen Risk phrases R21 Harmful in contact with skin R25 Toxic if swallowed R36/38 Irritating to eyes and skin R43 May cause sensitisation by skin contact R45 - May cause cancer 2. Chemical hazard SDS Harmful Risk phrases R10 Flammable R22 Harmful if swallowed R38 - Irritating to skin 3. Chemical hazard APS Oxidising / Corrosive Risk phrases R22 Harmful if swallowed R36/37/38 Irritating to eyes and respiratory system and skin R42/43 May cause sensitisation by inhalation and skin contact R8 - Contact with combustible material may cause fire 4. Chemical hazard Flammable Risk phrases R11 Highly flammable R20 Harmful by inhalation R22 - Harmful if swallowed 5. Spillage of hazardous substances due to leaking apparatus 6. Cuts from glass plates WRITTEN BY CHECKED BY AUTHORIZED BY NAME (signed) Ashman Group Lynn Herd Phillip Dickson DATE < st December th January 2007 Distributed To: GLP Master File / GLP Lab File

2 Page: 2 of 6 RISK ASSESSMENT 1. The risk of contamination from chemicals is low providing the appropriate personal protective equipment is used. 2. Risk of spillage is moderate given the type of apparatus used and its age. Chemicals may leak during casting due to poor seals or when the apparatus is assembled incorrectly. 3. The risk of cuts from the glass plates is low. RISK CONTROL 1. Acrylamide Safety phrases S45 In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible) S53 - Avoid exposure - obtain special instructions before use 2. SDS Safety phrases S22 Do not breathe dust S25 Avoid contact with eyes S36 Wear suitable protective clothing S51 - Use only in well ventilated areas 3. APS Safety phrases S17 Keep away from combustible material S22 Do not breathe dust S25 Avoid contact with eyes S36 - Wear suitable protective clothing 4. Temed Safety phrases S1 Keep locked up S16 Keep away from sources of ignition - No Smoking! S23 Do not breathe gas/fumes/vapour/spray S25 - Avoid contact with eyes 5. Training and practice in assembling the apparatus is necessary to reduce the occurrence of spills. 6. A disposable piece of benchcoat should be laid on the gel-casting area prior to casting and disposed into the contaminated waste when gel casting has been completed.

3 Page: 3 of 6 7. Any chipped or cracked glass plates should be disposed into a Sharps container. 8. Ensure that glass plates are held carefully and no undo pressure is exerted to cause breakage of glass. 9. Wear appropriate PPE particulate mask when weighing out powdered SDS; safety glasses; nitrile gloves and lab gown. 10. Training should be provided by personnel experienced in this procedure. 11. Training should be obtained in Laboratory Safety 2. Purpose: 2.1. To prepare SDS Polyacrylamide gels for electrophoresis of proteins to achieve protein separation. 3. Equipment: 3.1. Glass plate (s) 3.2. Aluminium backing(s) 3.3. spacers 3.4. combs 3.5. gel caster 3.6. pipettors 4. Materials: 4.1. Kim wipes 5. Reagents: 5.1. (Buffer A) g/100 ml of Milli Q - bring down ph with 10 M HC M Tris ph 6.8 (Buffer B) g/100 ml of Milli Q - bring down ph with 10 M HC1 (filter through um filter and store buffers at 4 C) 5.3. /Bis (3:1) (BioRad cat no or Applichem cat no. A1672,1000) % Ammonium persulphate 10% w/v in Milli Q water, stored at -20 C in 500 ul aliquots

4 Page: 4 of (Sigma: cat no T-9281 store at 4 C) 5.6. (10% w/v in milliq water stored at RT) 5.7. SDS PAGE ELECTRODE BUFFER - (for 5 Litres) g Tris, g glycine and 5 g SDS (use mask when weighing this out) DO NOT ph THIS SOLUTION 5.8. Separating Gel: 6% 5 ml 10 ml 15 ml 20 ml 25 ml 30 ml 40 ml 50 ml % 5 ml 10 ml 15 ml 20 ml 25 ml 30 ml 40 ml 50 ml % 5 ml 10 ml 15 ml 20 ml 25 ml 30 ml 40 ml 50 ml % 5 ml 10 ml 15 ml 20 ml 25 ml 30 ml 40 ml 50 ml % 5 ml 10 ml 15 ml 20 ml 25 ml 30 ml 40 ml 50 ml

5 Page: 5 of Stacking Buffer: 2 ml 4 ml 10 ml 12 ml 15 ml 30% acrylamide M Tris (ph 6.8) Set Up: 6.1. Ensure that you have read and understood the Safety Precautions (Section 7 of this SOP) prior to commencing this procedure 6.2. Clean gel apparatus with ethanol and kimwipes.. Place a piece of bench-coat onto bench where gels are to be prepared. 7. Safety Procedures: 7.1. Good laboratory techniques are to be used at all times Wear gloves throughout this procedure unpolymerised acrylamide is extremely toxic. 8. Method: 8.1. Place glass plate, spacers and backing together and made sure that they are flush Firmly holding the top centre with thumb and fore-finger, placed into "Hannibal Lector" device Screw down firmly but not too tightly using the 6 screws and then place in Gel Caster Insert black plastic knobs at both sides of caster with the longer arms pointing downwards, then simultaneously rotate them upwards 180 degrees Add water (from water bottle) to the gel space any leaking should be observed in 1-2 minutes. Empty and drain water Push comb in all the way and then mark on glass about 0.8cm down from bottom of teeth with a texta (this is to mark where to fill with separating gel). Remove comb.

6 Page: 6 of Prepare separating gel (5 mls for a mighty small gel) 8.8. Add APS and last just before pouring - mix and pour into gel-space using 5 ml pipette down the sides (inside) of the plates Immediately add 500 ul of Milli Q water Gel takes about minutes to set Once set, pour off water. Drain. Dab off excess with Whatman paper Make sure comb is in position - pushed in most of the way - leaving a small amount of space to fit pipette for loading stacking gel Use high quality acrylamide (Bio-Rad) Add stacking gel (2ml for a small gel) Again APS and should be added last just before pouring. Use P1000 pipette After stacking gel has set, carefully remove comb and transfer gel to gel tank in SDS PAGE Electrode Buffer. 9. Maintenance: 9.1. Polyacrylamide gels can be covered with foil and stored refrigerated overnight prior to using. Comb should be left in, or top of gel covered with Electrode buffer. 10. Shutdown: 1. Excess acrylamide should be allowed to set and then disposed into contaminated waste bag. 1. Dispose of bench-coat into contaminated waste. 11. Change History: Issue Number: 1st Issue Date Issued: 30 th January Issue Number: Date Issued: Reason for Change: