OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
STGCL.SWP.75.1
School/ Divisional Unit
St George Clinical School
Initial Issue date
15/04/10
Current version
1.0
Current Version
Issue date:
15/04/2010
Next review date
15/04/2012
The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist in the completion of this
form.
Safe Work Procedure Title and basic description
Title: RhoA activation assay
Description: This technique is used to measure RhoA activation spectrophotometrically.
Associated risk assessment title and location: STGCL.RA.75.1 RhoA activation assay
Describe the activity or process
This SWP outlines the correct procedure to be followed for cytotoxic assay. Read the Risk
Assessment attached and the relevant MSDSs before carrying out this procedure.
Procedures:
Collection of lysates:
i.
Culture media of cells grown to ~50% confluence is replaced by Cisplatin (Sigma)
(alkylating agent) pre-dissolved in culture media, dimethyl sulphoxide (DMSO) (Sigma)
(universal solvent), gemcitabine (Eli Lilly) (antimetabolite) pre-dissolved in culture media,
paclitaxel (Sigma) (microtubule targeting agent) pre-dissolved in DMSO and tyrphostin
AG1478 (Sigma) (EGFR inhibitor) pre-dissolved in DMSO for 24 hours.
ii.
Aspirate off PBS.
iii.
Harvest cell lysates with a cell scraper.
iv.
Transfer lysates into the pre-labeled sample tubes on ice and immediately clarify by
centrifugation at 10,000 rpm, 4°C for 2 min.
v.
Lysate protein concentration is determined spectrophotometrically using Precision Red TM
Advanced protein assay by absorbance at 600nm.
vi.
The rest of the lysates are snap frozen, and stored at -80oC until further use.
RhoA activation assay:
i.
Take the Rho plate out of its bag. Gently peel up the seal from the strips and pull out the
number of strips required. Place strips in the extra strip holder provided, and place on ice.
ii.
Dissolve the powder in the wells with 100 μl ice cold water, keep plate on ice.
iii.
Thaw the snap frozen cell lysates in room temperature water bath. Immediately place on ice
after they are thawed.
___________________________________________________________________________________________________________
Page 1 of 4
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
Describe the activity or process
iv.
Add required amount of Lysis Buffer to respective tubes to equalize all lysate
concentration.
v.
Immediately aliquot sufficient lysate for assays into fresh ice cold microcentrifuge tubes.
vi.
Add an equal volume of ice-cold Binding Buffer to each tube. Keep on ice.
vii.
Completely remove the water from the microplate wells.
viii.
Put plate back on ice.
ix.
Immediately vortex each tube for 3-5 s on a high setting and return tubes to ice.
x.
Immediately add 50 μl of equalized cell lysate to duplicate or triplicate wells.
xi.
Pipette 50 μl of buffer blank control into duplicate wells.
xii.
Pipette 50 μl of RhoA positive control into duplicate wells.
xiii.
Immediately place the plate on a cold orbital microplate shaker (400 rpm recommended,
200 rpm minimum) at 4°C for exactly 30 min.
xiv.
After 30 min, flick out the solution from the wells and wash twice with Wash Buffer at room
temperature using a multi-channel pipettor.
xv.
Place plate on the bench.
xvi.
Immediately pipette room temperature Antigen Presenting Buffer into each well using a
multichannel pipettor and incubate at room temperature for exactly 2 min.
xvii.
Vigorously flick out the Antigen Presenting Buffer.
xviii.
Immediately wash the wells three times with room temperature Wash Buffer.
xix.
Add diluted anti-RhoA primary antibody to each well and leave the plate on the orbital
microplate shaker (400 rpm) at room temperature for 45 min.
xx.
Vigorously flick out the anti-RhoA primary antibody.
xxi.
Immediately wash the wells three times with room temperature Wash Buffer.
xxii.
Add diluted Secondary antibody to each well and leave the plate on a microplate shaker
(400rpm) at room temperature for 45 min.
xxiii.
Vigorously flick out the secondary antibody.
xxiv.
Wash the wells three times with 200 μl of Wash Buffer
xxv.
Pipette HRP detection reagent into each well and incubate at 37°C for 15 min.
xxvi.
Add HRP Stop Buffer.
xxvii.
Immediately read the signal by measuring absorbance at 490 nm using a microplate
spectrophotometer.
___________________________________________________________________________________________________________
Page 2 of 4
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
Equipments:
 Cell scraper for cell collection
 Class II Biological Safety Cabinets
 Ice buckets containing ice (it is useful to have a separate ice bucket for cell harvesting)
 1.5 ml microfuge tubes, ice cold
 Micropipettes
 Multichannel pipette
 Pippette aid
 Plate shaker
 5ml/ 10ml/ 25ml serological pipettes
 Tips
 Vortex
Chemicals:
 Wash Buffer
 Precision RedTM Advanced Protein Assay Reagent
 Water, 30 ml, ice cold
 Binding Buffer, ice cold
 HRP Stop Solution, acid added
 Lysis Buffer, ice cold with protease inhibitors
 PBS, ice cold
 Rho Control Protein, resuspended in Lysis Buffer on ice
 Antibody Dilution Buffer
 Liquid nitrogen for lysates snap freezing
PPCE:
 Gloves
 Gown
List potential hazards and risk controls including
specific precautions required

Refer to relevant risk assessment form.
List emergency shutdown instructions
List clean up and waste disposal requirements
Waste disposal:
i.
Culture media is treated with 1% hypochloride (total volume) and leave treatment for at least 30 minutes before
transfer to the culture waste container
___________________________________________________________________________________________________________
Page 3 of 4
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007
List legislation, standards and codes of practice used
in the development of the SWP
 MSDS
 OH & S policies
Supervisory approval, training, and review
Supervisor: Peter Galettis
Signature:
Plant custodian: Peter Galettis
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
Researcher to attend trainings as follow:

Induction program

Laboratory Safety Awareness

PC2 Bio Training

Hazardous Substances

OHS Awareness for Employees

St George Hospital Annual Mandatory Training
SWP review date: 15/04/2012
Responsibility for SWP review: Steven Lim
___________________________________________________________________________________________________________
Page 4 of 4
Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007