Standard Operating Procedure
Nitrocellulose
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Laboratory Safety Manual and Chemical Hygiene Plan.
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Date SOP was approved by PI/lab supervisor: / Click here to enter a date.
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Type of SOP: ☐Process ☒Hazardous Chemical ☐ Hazardous Class
Purpose
Nitrocellulose is used commonly in membrane in protein immunoblot experiments. It can also be used to manufacture explosives and propellants. Membranes are disposed as hazardous waste. Nitrocellulose membranes can be purchased with either 0.45um or 0.2um pore size. The size of the membrane varies depending on the type of blotting experiment. Proper PPE must be worn when handling nitrocellulose membranes.
Physical & Chemical Properties/Definition of Chemical Group
CAS#: 9004-70-0
Class: Potentially explosive chemical
Molecular Formula:
Form (physical state): Click here to enter text.
Color: Click here to enter text.
Boiling point: No data
Potential Hazards/Toxicity
EMERGENCY OVERVIEW: Explosive when dry. Risk of explosion by shock, friction, fire or other sources of ignition. Harmful to aquatic organisms.
Potential Health Effects:
Swallowed: Although ingestion is not thought to produce harmful effects, the material may still be damaging to the health of the individual following ingestion, especially where pre-existing organ (e.g. liver, kidney) damage is evident. Considered an unlikely route of entry in commercial/industrial environments. The substance and/or its metabolites may bind to hemoglobin inhibiting normal uptake of oxygen. This condition, known as "methemoglobinemia", is a form of oxygen starvation (anoxia).
As a high molecular weight material, nitrocellulose would be expected to pass through gastrointestinal tract with little change / absorption on single acute exposure. Occasionally accumulation of the solid material within the alimentary tract may result in formation of a bezoar (concretion), producing discomfort.
Eye: Although the material is not thought to be an irritant, direct contact with the eye may produce transient discomfort characterized by tearing or conjunctival redness (as with windburn). The dust may produce eye discomfort causing smarting, pain and redness.
Skin: The material is not thought to produce adverse health effects or skin irritation following contact (as classified using animal models). Nevertheless, good hygiene practice requires that exposure be kept to a minimum and that suitable gloves be used in an occupational setting.
Inhaled: The material is not thought to produce adverse health effects or irritation of the respiratory tract (as classified using animal models). Nevertheless, good hygiene practice requires that exposure be kept to a minimum and that suitable control measures be used in an occupational setting.
Chronic health effects: Principal routes of exposure are by accidental skin and eye contact and inhalation of generated dusts. This material contains a substantial amount of polymer considered to be of low concern. These are classified under having MWs of between 1000 Da to 10000 Da with less than 25% of molecules with MWS under 1000 Da and less than 10% under 500; or having a molecular weight average of over 10000 Da.
Personal Protective Equipment (PPE)
Respirator Protection
Use if working with particulates. (AS/NZS 1716 & 1715, EN 143:2000 & 149:2001, ANSI Z88 or national equivalent)
Respirators should be used only under any of the following circumstances:
· As a last line of defense (i.e., after engineering and administrative controls have been exhausted).
· When Permissible Exposure Limit (PEL) has exceeded or when there is a possibility that PEL will be exceeded.
· Regulations require the use of a respirator.
· An employer requires the use of a respirator.
· There is potential for harmful exposure due to an atmospheric contaminant (in the absence of PEL)
· As PPE in the event of a chemical spill clean-up process
Lab personnel intending to use/wear a respirator mask must be trained and fit-tested by EH&S. This is a regulatory requirement. (https://www.ehs.ucla.edu/ep/ih/resp)
Hand Protection
Rubber gloves are recommended.
NOTE: Consult with your preferred glove manufacturer to ensure that the gloves you plan on using are compatible with Nitrocellulose.
Refer to glove selection chart from the links below:
http://www.ansellpro.com/download/Ansell_8thEditionChemicalResistanceGuide.pdf
OR
http://www.allsafetyproducts.com/glove-selection-chart-chemical-breakthrough-ratings.html
OR
http://www.showabestglove.com/site/default.aspx
OR
http://www.mapaglove.com/
Eye Protection
Wear safety glasses with side shields.
Skin and Body Protection
Lab coats should be worn. These laboratory coats must be appropriately sized for the individual and be buttoned to their full length. Laboratory coat sleeves must be of a sufficient length to prevent skin exposure while wearing gloves. Full length pants and close-toed shoes must be worn at all times by all individuals that are occupying the laboratory area. The area of skin between the shoe and ankle should not be exposed.
Manufacture may require: Non-static flame retardant treated clothing and access to deluge safety shower
Hygiene Measures
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Engineering Controls
General exhaust is adequate under normal operating conditions. Local exhaust ventilation may be required in specific circumstances.
First Aid Procedures
If inhaled
If fumes or combustion products are inhaled remove from contaminated area. Lay patient down. Keep warm and rested.
In case of skin contact
Immediately remove all contaminated clothing, including footwear. Flush skin and hair with running water (and soap if available).
In case of eye contact
Immediately hold eyelids apart and flush the eye continuously with running water. Ensure complete irrigation of the eye by keeping eyelids apart and away from eye and moving the eyelids by occasionally lifting the upper and lower lids.
If swallowed
IF SWALLOWED, REFER FOR MEDICAL ATTENTION, WHERE POSSIBLE, WITHOUT DELAY.
Special Handling and Storage Requirements
PROCEDURE FOR HANDLING
Handle gently. Use good occupational work practice. Observe Manufacturer's storing and handling recommendations. Avoid formation of dust and aerosols. Provide appropriate exhaust ventilation at places where dust is formed. Keep away from sources of ignition. Take measures to prevent the build-up of electrostatic charge.
RECOMMENDED STORAGE METHODS
All packaging for Class 1 Goods shall be in accordance with the requirements of the relevant Code for the transport of Dangerous Goods.
STORAGE REQUIREMENTS
For solutions: Store drums on end and invert them regularly (at least monthly) to avoid separation of the desensitizing liquid. Keep dampened. Do NOT allow to dry. Store cases in a well-ventilated magazine licensed for the appropriate Class, Division and Compatibility Group. Rotate stock to prevent ageing. Use on FIFO (first in-first out) basis. DO NOT store as dry material. When nitrocellulose is wetted with water or phlegmatiser avoid storage at temperature greater than 40C.
For membranes: Keep container tightly closed in a dry and well-ventilated place. Store in cool place.
Spill and Accident Procedure
Chemical Spill Dial 911 and x59797
Spill – Help contaminated or injured persons. Evacuate the spill area. Avoid breathing vapors utilizing a self-contained breathing apparatus. Eliminate sources of ignition if the chemical is flammable. If possible, confine the spill to a small area using a spill kit or absorbent material. Vacuum or sweep up material and place into a suitable disposal container. Avoid runoff into storm sewers and ditches which lead to waterways. Clean up spills immediately, observing precautions in the Protective Equipment section. Avoid generating dusty conditions. Decontaminate trace cyanide in the spill area with a strong sodium or calcium hypochlorite solution and flush waste to a holding area for potassium removal. Provide ventilation. Prevent spreading of vapors through sewers, ventilation systems and confined areas. Evacuate unnecessary personnel. Keep others from entering contaminated area (e.g., use caution tape, barriers, etc.)
Small (<1 L) – If you have training, you may assist in the clean-up effort. Use appropriate personal protective equipment and clean-up material for chemical spilled. Double bag spill waste in clear plastic bags, label and take to the next chemical waste pick-up.
Large (>1 L) – Dial 911 (or 310-825-1491 from cell phone) and EH&S at x59797 for assistance.
Chemical Spill on Body or Clothes – Remove clothing and rinse body thoroughly in emergency shower for at least 15 minutes. Seek medical attention. Notify supervisor and EH&S at x59797 immediately.
Chemical Splash Into Eyes – Immediately rinse eyeball and inner surface of eyelid with water from the emergency eyewash station for 15 minutes by forcibly holding the eye open. Seek medical attention. Notify supervisor and EH&S at x59797 immediately.
Medical Emergency Dial 911 or x52111
Life Threatening Emergency, After Hours, Weekends And Holidays – Dial 911 (or 310-825-1491 from cell phone) or contact the Ronald Reagan UCLA Medical Center (emergency room) directly at x52111 (located at 757 Westwood Plaza, enter from Gayley Avenue). Note: All serious injuries must be reported to EH&S at x59797 within 8 hours.
Non-Life Threatening Emergency – Go to the Occupational Health Facility (OHF), x56771, CHS room 67-120 (This is on the 6th floor, 7th corridor, room 120. Enter through the School of Dentistry on Tiverton Drive and proceed to the “O” elevator to the 6th floor.)Hours: M - F, 7:30 a.m. to 4:30 p.m. At all other times report to Ronald Regan UCLA Medical Center (emergency room) at x52111. Note: All serious injuries must be reported to EH&S at x59797 within 8 hours.
Needle stick/puncture exposure (as applicable to chemical handling procedure) – Wash the affected area with antiseptic soap and warm water for 15 minutes. For mucous membrane exposure, flush the affected area for 15 minutes using an eyewash station. Page the needle stick nurse by dialing 231 from a campus phone, enter 93333 when prompted and then enter your extension. Hours: M – F, 8:00 a.m. to 4:00 p.m. At all other times report to Ronald Regan UCLA Medical Center (emergency room) at x52111. Note: All needle stick/puncture exposures must be reported to EH&S at x59797 within 8 hours.
Decontamination/Waste Disposal Procedure
All waste must be handled in accordance with local, state and federal regulations. Explosives which are surplus, deteriorated or considered unsafe for transport, storage or use shall be disposed of (Contact if you have questions about disposal) and the statutory authorities shall be notified. Explosives must not be thrown away, buried, discarded or placed with garbage.
General hazardous waste disposal guidelines:
Label Waste
· Affix an on-line hazardous waste tag on all waste containers using the WASTe Online Tag Program https://ehs.ucop.edu/waste as soon as the first drop of waste is added to the container
Store Waste
· Store hazardous waste in closed containers, in secondary containment and in a designated location
· Double-bag dry waste using transparent bags https://www.ehs.ucla.edu/hazwaste/management/containers
· Waste must be under the control of the person generating & disposing of it
Dispose of Waste
· Dispose of regularly generated chemical waste within 90 days
· Call EH&S at x61887 for questions
· Empty Containers
o Dispose as hazardous waste if it once held extremely hazardous waste (irrespective of the container size) https://www.ehs.ucla.edu/hazwaste/types/extremely-hazardous
o Consult waste pick-up schedule
https://www.ehs.ucla.edu/hazwaste/management/pick-ups
Prepare for transport to pick-up location
§ Check on-line waste tag
§ Write date of pick-up on the waste tag
§ Use secondary containment
Safety Data Sheet (SDS) Location
Online SDS can be accessed at http://msds.ehs.ucla.edu.
Protocol/Procedure
Quantities covered by this SOP
1-2 membrane(s)
Conditions covered by this SOP
4 ºC - 25 ºC
Protein Immunoblot Experiments
Nitrocellulose (NC) membranes are placed in a gel tray and wetted with water and then placed in electrophoresis western blot transfer buffer. A typical membrane will be cut to the gel size of 3” x 4”. PAGE gels placed on a pre-wetted nitrocellulose membrane and subsequently subjected to electrophoresis to transfer proteins from the gel onto the membrane. NC membranes are then blocked with protein containing solution and exposed to antibodies via a standard western blot and immune-stain protocol such as the one shown below.
ECL: Western Blot
1. Run AU or SDS PAGE
2. Wet Nitrocellulose membrane in water and then incubate in transfer buffer for 10 min. Transfer buffer
· AU: 0.7% acetic acid, 10% MeOH
· SDS: .05M Naborate pH 9, 20% MeOH, 1% SDS
3. Set up W. blot and transfer at 0.18A
· AU: 20 min
· SDS: 1 hour
4. Wash TBS 5 min
5. Block in Superblock or 0.75% Blotto/TBS 30 min 37oC
6. Incubate in 1st antibody diluted in Superblock 1 hr at RT
· HBD-1: 1-10,000
· HBD-2: 1-20,000
7. Wash briefly in dH20 then 5 min in TTBS*
8. Change trays and wash 4-6 times TTBS* 8-10 min each
9. Incubate in 2nd Ab; GAR-HRPO diluted 1:20,000 in Superblock
10. Wash as in steps 8 & 9
11. Incubate blots for 5 min in substrate solution (mix equal vol of each sol'n)
12. Place in sheet protector to read in chemidoc system
13. Dispose membrane as hazardous waste
NOTE
Any deviation from this SOP requires approval from PI.
Documentation of Training (signature of all users is required)
· Prior to conducting any work with Nitrocellulose, designated personnel must provide training to his/her laboratory personnel specific to the hazards involved in working with this substance, work area decontamination, and emergency procedures.
· The Principal Investigator must provide his/her laboratory personnel with a copy of this SOP and a copy of the SDS provided by the manufacturer.
· The Principal Investigator must ensure that his/her laboratory personnel have attended appropriate laboratory safety training or refresher training within the last one year.
Principal Investigator SOP Approval
Print name ______Signature______