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= All buffers must be filtered or otherwise rendered particle-free. pH 2-10 can be used Sterile PBS can be used as a buffer. A maximum of 2ml can be run throu..."

2021-02-01T19:53:05Z

Created page with "= Notes = *All buffers must be filtered or otherwise rendered particle-free. *pH 2-10 can be used *Sterile PBS can be used as a buffer. *A maximum of 2ml can be run throu..."

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= Notes =

*All buffers must be filtered or otherwise rendered particle-free.
*pH 2-10 can be used
*Sterile PBS can be used as a buffer.
*A maximum of 2ml can be run through the column at any one time
*Sample must be filtered or otherwise rendered particle-free
*Sample should be as concentrated as possible, 1mg/ml or better
*Roomkey: 3524 

= Protocol =

===== Column Preparation: =====

#Use Sephacryl S100 column 
#Remove top cap, flood top surface with buffer (PBS or other) 
#Place column in clamps 
#Take top port tube, and place outlet into a waste container. 
#Take inlet tube 'A', dip in dH2O, and then immerse in a minimum of 300ml buffer 
#Run pump wash program: 
#*Turn bottom knob to “pump wash”, press OK on dial 
#*~20ml of fluid will run through tubing, takes ~2min 
#While wash cycle is running, remove bottom plug from column and attach outlet tube 
#When wash is done, run pump at slow speed (~0.5ml/min) 
#*Turn bottom knob to “Set Flow Rate”, press OK on dial 
#*Turn bottom knob to 0.5ml/min, press OK on dial 
#Once drops emerge from inlet tube, attach to upper port of column 
#*A drop from the inlet tube should be touched to the layer of buffer on the column to avoid air bubbles 
#Turn off flow 
#*Turn bottom knob to “End”, press OK on dial 

===== Column Wash: =====

#Start Computer:
#*UID: fplc_user 
#*PW: 4020 
#Start “Unicorn” program on computer 
#*UID: Moshe 
#*PW: 123456 
#Select “System Control” tab on taskbar 
#Under 'File' menu select “Run” 
#Select the “Wash S100” program 
#Click “Next” until window with “start” appears, click “Start”
#Check column depositor to ensure buffer is running into the waste container. 
#*If no flow occurs after a few minutes there is likely an air bubble in the system. This should also cause an over-pressure alarm and automatic shutdown of the wash column program.
#Allow wash to proceed. Wash requires 2 column volumes (240ml total), and runs at 0.5ml/min (8 hrs total run time).
#Column is now ready for sample

===== Sample Addition and Separation: =====

#Filter sterilize digest, using a small-volume, non-protein binding filter.
#*Alternatively, spin in a bench-top minifuge at max speed, 4oC for 20min.
#Fill columns A-G of the collection tray with capless 1.5-2ml eppindorf tubes.
#Place collection tray on pedestal; slot lines up with pin.
#Stop flow, if flow did not terminate on its own.
#Top up buffer, a minimum of 180ml is needed
#Flush needle (located on top of control boxes) with a min of 3ml buffer
#Inject ~3ml buffer into sample port on pump.
#Draw sample into needle, using a clean 1ml syringe
#Inject sample into sample port, leave needle in port as withdrawing it may create a vacuum
#Start unicorn, run the “Ron S100” program 
#*Flow rate should be 0.25ml/min 
#*Load volume should be 1.2ml 
#*Collection Volume should be 1.2ml/tube 
#*Elution volume should be 1.5 column volumes (180ml) 
#Click “next” on the box until you reach the screen with the “Start” button
#Type in a file name for the trace file which is created
#Click start. Run will start. Monitor fraction collector to ensure that fractions are landing in the collection tubes.
#Run will take ~10hrs.
#After run, use UV trace and/or western blotting to identify fractions containing the sample.
#Western blot under non-reducing conditions to ID F(ab')2 containing fractions. Pool & concentrate for final use.

===== Running Another Sample: =====

#Re-load sample collection tray
#Inject 3ml of buffer into sample port, as done previously
#Inject sample into sample port, as done previously
#Run sample using “Ron S100” protocol 

===== Preparing Column for Storage: =====

#Remove input tube from top of column.
#Immerse inlet tube 'A' in at least 300ml of 20% ethanol in ddH2O.
#Clear input tube an pump, using the pump clear protocol described in steps 4-10 of “Column Preparation”
#Clean the column, as per the “Column Wash” portion of this protocol, using 20% ethanol in place of buffer.
#Once wash cycle is complete, remove input/output tubes, cap column, and store column behind the column stand.

Fab Purification by FPLC - Revision history

Admin: Created page with "= Notes = *All buffers must be filtered or otherwise rendered particle-free. *pH 2-10 can be used *Sterile PBS can be used as a buffer. *A maximum of 2ml can be run throu..."

Admin: Created page with "= Notes
= All buffers must be filtered or otherwise rendered particle-free. pH 2-10 can be used Sterile PBS can be used as a buffer. A maximum of 2ml can be run throu..."