Risk assessment Copy of Westernblot onk. Dabrosin [swe]

2011-10-12 09:03
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Risk assessment
Copy of Westernblot onk. Dabrosin[swe]
Produced 2011-10-11 By Helena Tegehed-Dahli at Onkologi
Dabrosin.
Final risk assessment of the method
High risk
1. State the premises in which the activity is taking place
2. Description of activity
ECL Detektion Kit: ECL Western blotting system, Ge-healthcare #RPN2108
Material and method
ECL Detektion Kit: ECL Western blotting system, Ge-healthcare #RPN2108
(Contains substrate and HRP conjugated secondary antibody, anti mouse and anti rabbit)
Perfect Protein Western Markers: Novagen: Western marker #69959
Novagen: S-protein HRP-conjugate #69047
Gel: Tris/HCl 15%, Bio Rad #456-1083
Running buffer: Tris/Glycin/SDS, Bio Rad # 161-0732
Sample buffer: Laemmli sample Buffer , Bio Rad #161-0737
2-Mercaptoethanol: Sigma, # M7154-25ml.
Fiber pads: 11 x 8 cm, Bio Rad #1703933
Membran: PVDF/filter membrane, Bio Rad, #162-0238
Bio-Ice Cooling unit: Bio Rad #170-3934
Transfer buffer: 10X Tris/Glycin buffert (Bio Rad#161-0734) 100 ml
dd H2O 700 ml
Methanol 200 ml
- Buffer has to be chilled before use!!
Wash buffer: 0.05 M Trizma pH 7.5 (Sigma # 93372-100G), 0,15 M NaCl,
1 l buffer: Trizma 7.5 g
NaCl 8.77 g
Block buffer: 5% non fat dry milk, Bio Rad # 170-6404, in Wash buffer with 0.1% Tween
100 ml buffer Non fat dry milk 5 g
Tween 20 100 ul
Wash buffer 100 ml
Electroforesis (reduced with ß-mercaptoethanol)
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Mix Running buffer (10x Tris/Glycin/SDS) in deionised H2O
Place the gel (Tris/HCl 4-15%) in the electrophoresis container and fill with Running buffer
Solve protein sample in Laemmli sample buffer containing 5% 2-Mercaptoethanol.
Inkubate samples 5 min at +95ºC
Load samples and standard on the gel
• Run at 200V 60 mA
• Stop the electrophoresis when all samples are at the bottom of the gel
Western Blott
Transfer
• Make sure that the transfer buffer is cold and that you have a frozen ice block
• Equilibrate the gel for 15 min. in transfer buffer
• Pour chilled transfer buffer into each compartment of the gel/blot assembly tray
• Pre wet the PVDF membrane in 100% MeOH
• Then place the membrane in the front/small compartment of the tray containing transfer buffer, and let it soak.
• Place the cassette in the back/large compartment of the tray: Open the cassette so
the red side with the handle is vertical (anode) and the black side (cathode) is
laying horizontal and submerged in transfer buffer.
• Place a fiber pad on top of the black side of the cassette, submerged in buffer. Push
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• Place a fiber pad on top of the black side of the cassette, submerged in buffer. Push
on the fiber pad with gloved finger tips to thoroughly soak the pad.
• Place a piece of filter paper on top of the fiber pad (it will wet immediately).
• Gently place the pre-equilibrated gel on top of the filter paper.
• Take the membrane from the front compartment and place it on top of the gel taking
care not to trap any air bubbles. The membrane should not be moved or adjusted
once it touches the gel because this can cause data ghost prints and artefacts. If you
feel that you must adjust the membrane placement, use a fresh pre-wetted membrane. Cut one of the corners of the membrane so you know where
the protein side is after transfer.
• Place a piece of filter paper on top of the membrane.
• Run the roller gently over the top of the filter paper to remove any air bubbles trapped in the sandwich.
• Wet a second fiber pad in the front compartment of the tray (where the membrane was
soaking) using glowed finger tips to completely saturate the pad with transfer buffer.
Then place the wet fibre pad on top of the second filter paper.
• Lower the clamp-side of the cassette, and lock in the closed position.
• Move the locked cassette into the groove in the blotter tank, aligning the red side of
the card with the red electrode. Make sure the magnetic stirrer is free to move and put the ice block in place.
• After cassette is in place, add the remaining transfer buffer to the fill level
marked on the tank.
• Put on the lid, plug the cables into the power supply, and run the blot.
• Transfer at a constant voltage 100V 250 mA
• Upon completion of the run, disassemble the blotting sandwich and remove the
membrane for development. Clean the cell, fiber pads, and cassettes with multiple
rinses of deionized water.
Washing and blocking
• Membrane is transferred to a container with Washbuffer/0.05% Tween, the protein side of the membrane facing upwards.
• Decant the wash buffer and add blocking buffer, incubate 90 min at RT on a gentle rocker.
• Rinse the membrane ones in wash-buffer/0.05% Tween, then wash 3 x 15 min.
Detection
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Dilute primary antibody stock in block buffer
Transfer membrane to a plastic film and seal the sides so it becomes like a bag.
Add the primary antibody solution into the bag with the membrane.
Incubate 2 hours at RT or ON at +4C on a rocker.
Rinse the membrane ones in wash-buffer/0.05% Tween, then 2-3 times for 15 min.
Dilute secondary antibody in block buffer, also dilute the S-protein conjugate in the same solution.
Washed membrane is then placed in a plastic film as previously described and secondary antibody-solution is added.
Incubate at RT for 1 hour on a rocker.
Wash membrane, 3 x 5 min in wash buffer/0.05% Tween.
Turn of the light and work in dark.
Mix equal volumes of detection solution 1 and 2 ( 2 + 2 ml/membrane)
Place a piece of parafilm on the lab bench – pre wet with H2O.
Lift the membrane from the wash buffer; take away remaining water by placing the edge of the membrane on a dry paper towel.
Place the membrane on the Parafilm with the protein side facing up.
Gently pipette the detection solution on the membrane so it covers the hole surface
Incubate for 1 min.
Lift the membrane against a paper towel to get rid of the reagent.
Put the membrane in a plastic film and place in a cassette to protect it from light.
For development go to level 12 at the Cell-biology Department and use the CCD camera.
3. Products
Product name
CAS-nr
Quant/ Form
Konc
Volume Hazard symbol
Comments
2-Mercaptoethanol
CLP-legislation
H301 , H310 , H315 , H317 , H318 , H330 , H400 , H410
P260 , P273 , P280 , P284 , P302 + P350 , P302 + P352 , P304 + P340 , P305 +
P351 + P338 , P309 + P310
older legislation
R23/24/25 , R38 , R41 , R43 , R50/53
S26 , S36/37/39 , S45 , S60 , S61
60-24-2
Solution
startprodukt
Methanol 61 - 100%
CLP-legislation
H225 , H301 , H311 , H331 , H370
P210 , P233 , P280 , P302 + P352 , P309 , P310
older legislation
R11 , R23/24/25 , R39/23/24/25
S16 , S36/37 , S45 , S7
Lists
Restricted Substances Database
Note H
Organic compounds
Tween 20
Risk assessment : Copy of Westernblot onk. Dabrosin
67-56-1
9005-64-5
99,7 % Liquide
startprodukt
Liquide
startprodukt
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Health statements
H370 Causes damage to organs .
Precautionary statements
P310 Immediately call a POISON CENTER or doctor/physician.
Risk phrases
R11
Highly flammable.
R23/24/25
Toxic by inhalation, in contact with skin and if swallowed.
R38
Irritating to skin.
R39/23/24/25 Toxic: Danger of very serious irreversible effects through inhalation, in contact with skin and if swallowed.
R41
Risk of serious damage to eyes.
R43
May cause sensitisation by skin contact.
R50/53
Very toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment.
Safety advice phrases
S16
Keep away from sources of ignition - No smoking
S26
In case of contact with eyes, rinse immediately with plenty of water and seek medical advice
S36/37
Wear suitable protective clothing and gloves
S36/37/39 Wear suitable protective clothing, gloves and eye/face protection
S45
In case of accident or if you feel unwell, seek medical advise immediately. Show the label where possible
S60
This material and its container must be disposed of as hazardous waste
S61
Avoid release to the environment. Refer to special instructions/Safety data sheets
S7
Keep container tightly closed
Hazard
T Toxic
N Dangerous for the environment
F Highly flammble
4. Risk category
b: High risk
5. Level of exposure
High
6. Ventilation
Level of protection 2 - ventilated hood
7. Risk codes
T
F
N
Toxic
Highly flammble
Dangerous for
the environment
8. Comments to risk codes
9. Emergency equipment
first aid kit , emergency shower , eye shower , fire-extinguisher carbonic acid , fire blanket , absorbing substance
10. Personal protective equipment
protective glasses , protective gloves , protective clothing
11. premises
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12. Protective signs
13. Waste
chemical waste
14. Hazardous actions
leakage , transference betw vessels , splash
15. Accidental readiness
FÖRSTA HJÄLPEN
- Vid inandning: Om det har andats in, flytta personen till frisk luft. Om andningen har upphört ge konstgjord andning Kontakta läkare.
- Vid hudkontakt:Tvätta med tvål och mycket vatten. För omedelbart patienten till sjukhus. Kontakta läkare.
- Vid ögonkontakt: Skölj grundligt med mycket vatten i minst 15 minuter och kontakta en läkare.
- Vid nedsväjning: Framkalla INTE kräkning. Ge aldrig någonting genom munnen till en medvetslös person. Skölj munnen med vatten. Kontakta
läkare.
BRANDBEKÄMPNINGSÅTGÄRDER
Lämpliga släckmedel:
- Vid små bränder använd släckmedel som t ex ånga, pulver eller koldioxid.
- Vid stora bränder använd vatten i möjligaste mån. Använd stora mängder distribuerade som dimma eller spray; annan vattenapplikation skulle
vara ineffektivt. Kyl ned alla behållare med stora mängder av vatten.
Särskild skyddsutrustning för brandbekämpningspersonal:
Använd tryckluftsmask om nödvändigt vid brandbekämpning.
Ytterligare information Använd finfördelat vatten för att kyla oöppnade behållare.
ÅTGÄRDER VID OAVSIKTLIGA UTSLÄPP
Personliga skyddsåtgärder: Använd andningsskydd. Undvik inandning av ånga/dimma/gas. Sörj för lämplig ventilation. Avlägsna alla
antändningskällor. Evakuera personal till säkra platser. Var aktsam för ångor som kan ansamlas och bilda explosiva koncentrationer. Ångor kan
ansamlas i lågt belägna områden.
Miljöskyddsåtgärder
Förhindra fortsatt läckage eller spill om det kan göras på ett säkert sätt. Förhindra utsläpp i avloppssystemet.
Metoder och material för inneslutning och rengöring:
Förvara och samla upp spill med ickebrännbart absorbentmaterial (t ex sand, jord, diatoméjord, vermiculit)
och placera i en behållare för vidare hantering som avfall enlig lokala / nationella regler
16. Risk of error during the assay
Minor
17. Describe the technical equipment
18. Describe hazardous actions
Iaktag försiktighet när metanol hämtas från kemikalie förrådet , använd helst en korg eller hink att transportera flaskan i. Blanda lösning med
metanol eller merkaptoetanol i dragskåp. Även pipettering av prover blandade med merkaptoetanol ska pipetteras i dragskåp.
19. Special instructions to other personel
20. Comments
Hantering av geler för elektrofores ska ske med handskar. Genomgång samt laborativt arbete för studenter sker alltid med utbildad personal.
21. Final risk assessment of the method
High risk
22. Comments to final risk assessment and additional risk reducing measurements
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Signature
Supervisor
Risk assessment : Copy of Westernblot onk. Dabrosin
Date