THE UNIVERSITY OF NEWCASTLE- DISCIPLINE OF MEDICAL BIOCHEMISTRY. STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP 047 MOD: 1st Issue Page: 1 of 7

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1 Page: 1 of 7 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 HAZARDS 1. Chemical hazard HCl for phing TBST (Corrosive) - Tris (Irritant) 2. Cuts use of razor blade 3. Burns from bag sealer RISK ASSESSMENT 1. The risk from the above hazards is low if the Controls are followed. RISK CONTROL 1. Wear lab coat, enclosed shoes and safety glasses when handling Solutions 2. Mop up spills using paper towels and dispose of in contaminated waste bag. 3. Dispose of all used reagents by flushing down the sink with excess water. 4. Use single sided razor blade only, cutting downwards away from the hands and body. 5. Store blade in plastic container and allow access to the blunt side only. 6. Keep fingers clear of bag when sealing plastic bag. 7. Training should be undertaken in General Laboratory Safety WRITTEN BY CHECKED BY AUTHORIZED BY NAME (signed) Lynn Herd Larisa Bobrovskaya Phil Dickson DATE 13 th October rd November rd November Distributed To: GLP Master File / GLP Lab File

2 Page: 2 of 7 2. Purpose: 2.1. Preparation of antigens (proteins) for electrochemiluminescence or NBT by binding to specific antibodies. 3. Equipment: 3.1. Plastic trays 3.2. Thick plastic suitable to be made into bags for addition of primary antibody 3.3. Bag sealer 3.4. Tweezers 3.5. Orbital shaker 3.6. Razor blade 4. Materials: 4.1. TBS (Tris Buffered Saline) 4.84 g TRIS g NaCl Make up to 4 litre with dd H 2 O. ph to 8.0 with conc HCl TBST (TBS/Tween 0.05%) 1 ml Tween 20 2 litre TBS % Milk Blocking Solution (1) 2.5 g Skim milk powder 50ml TBS Mix rapidly until dissolved using a magnetic stirrer. Make up fresh on the day of use. Store at 2-8 o C until used % Gelatin Blocking Solution (2) 1 g Gelatin powder 50 ml TBS

3 Page: 3 of 7 Mix rapidly until dissolved using a magnetic stirrer. Make up fresh on the day of use % BSA Blocking Solution (3) 2 g Bovine Serum Albumin 100 ml TBST Mix rapidly until dissolved using a magnetic stirrer. Make up fresh on the day of use Ponceau S Stain (0.5% in 1% Acetic Acid) 1 g Ponceau S stain 198 ml dd H 2 O 2 ml Acetic acid Dissolve Ponceau S in water, then add acid Primary antibody Make up to a suitable dilution for the specific antibody; add sodium azide as a preservative to a final concentration of 0.02%. Store at 4ºC. Antibodies may be used a number of times depending on the strength of the specific antibody used. Eg PP2Ac 1 o Antibody Make up to a dilution of 1/10000 in 2% BSA/TBST, add Na azide to f.c. 0.02% PP1c 1 o Antibody Make up to a dilution of 1/7000 in 2% BSA/TBST, add Na azide to f.c. 0.02% º Antibody Make up to appropriate dilution in one of TBST: 2%BSA/TBST; or 0.5% skim milk/tbst + Na azide Secondary antibody 1 for ECL (anti-mouse horseradish peroxidase at 1/5000) 3 µl anti-mouse horseradish peroxidase conjugated 15 ml TBS/T Make up on day of use. Store at 2-8 o C until used. Can be re-used once.

4 Page: 4 of 7 OR 4.9. Secondary antibody 2 for ECL (anti-rabbit horseradish peroxidase at 1/5000) 5 µl anti-rabbit horseradish peroxidase conjugated 15 ml TBS/T Make up on day of use. Store at 2-8 o C until used. Can be re-used once Secondary antibody 3 for NBT (anti-rabbit alkaline phosphatase at 1/5000) 3 µl anti-rabbit alkaline phosphatase conjugated 15 ml TBS/T Make up on day of use. Store at 2-8 o C until used. 5. Set Up: 5.1. Ensure that you have read and understood the Safety Precautions (Section 6 of this SOP) prior to commencing this procedure. 6. Safety Precautions: 6.1. Good laboratory techniques are to be used at all times. 7. Method: 7.1. Following Transfer of proteins onto ECL Hybond nitrocellulose or PVDF, cut the membrane into tracks or sections as required using a clean razor blade Always mark the bottom right (or left) corner of each section so that the orientation of the piece is identified. It is very easy to confuse the orientation without this procedure! 7.3. Specific proteins or antibodies will require the use of particular blocking agents. In general TBST by itself is sufficient as a Blocking agent. However if the blot is particularly dirty then skim milk, BSA or gelatin are other blocking agents suitable for use The following procedure can be used as a general guide for blocking, washing and incubation of antibody, however please be aware that the specific blocking agent required, the dilution of antibody and the number of times it can be used will vary according to the particular protein in which you are interested. It is usual to optimize the dilution of primary and secondary

5 Page: 5 of 7 antibodies and the incubation times for. Steps may be carried out at room temperature or at 4 o C, either in a powered fridge or cold room. It is recommended that where antibodies are left to incubate overnight this should take place at 4 o C Ponceau S staining for temporary visualization of proteins Stain the nitrocellulose quickly and lightly in Ponceau S to check the position of proteins and the success of the transfer Rinse the membrane in dd H 2 O to remove the Ponceau S. Three washes may be required Blocking Steps Using small plastic trays, soak the membrane in the first Blocking solution. TBST should be tried as the first Blocking agent. If it is not sufficient then use another. eg 5% Skim milk Block Shake on rocker for 60 minutes. *** NOTE *** Experiment may be paused at this point and the nitrocellulose membrane left in the Blockin solution in the coldroom overnight Tip off the blocking solution, and wash the membrane three times in TBS/Tween, soaking for minutes each wash If a second blocking solution is to be used, soak the membrane in the second Blocking solution, eg 2 % Gelatin and shake for 45 minutes Tip off the gelatin solution, and wash the membrane three times in TBS/Tween, soaking for minutes each wash Addition of Primary antibody Primary antibody can be added to membrane either directly into the plastic trays if sufficient antibody available, or by sealing membrane and antibody into small made-to-size plastic bags. The plastic bags have the advantage of requiring minimal Primary antibody volume If using bags, cut plastic to size to form bags.

6 Page: 6 of Place two nitrocellulose pieces together (if required), protein to the outside. Place on the plastic and then fold plastic over Seal sides of bags, using a double line of seal Add the Primary antibody to the bag 7-8 mls per bag, or less if only small membrane pieces are used Massage out the bubbles, and excess air Seal the top of the bag, again using a double line of seal Place on the rocker in the cold room and leave overnight, or on the rocker in the lab for 1-4 hours if continuing the western blot the same day Addition of Secondary antibody (conjugated with Horse-radish peroxidase) Recover the Primary antibody, and return it to the fridge until next use Wash membrane 3 times in TBS/T for minutes each wash Incubate with Secondary antibody in plastic tray or sealed bags on rocking platform at room temperature for 60 minutes Discard Secondary antibody after use Wash 3 times in TBS/T for minutes each wash Proceed to either ECL or NBT. 8. Maintenance: 8.1. Mark on the primary antibody bottle each time that it has been used. 9. Shutdown: 9.1. Rinse plastic trays in water, then deionized water after use Dispose of blocking solutions and secondary antibody down the sink with plenty of water Acrylamide gel should be disposed of as contaminated waste, or may be stained and dried to ascertain the success of the transfer. 10. Change History: Issue Number: 1st Issue Date Issued:

7 Page: 7 of Issue Number: Date Issued: Reason for Change:

8 STANDARD OPERATING PROCEDURE PROCEDURE NO: GLP Page: 8 of BATCH RECORD Page: 1 of Batch Record Completed By: Date: / / Countersigned: