View source for Fab Purification by FPLC

= Notes<br> =

*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<br>

= Protocol<br> =

===== Column Preparation:<br> =====

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

===== Column Wash:<br> =====

#Start Computer: 
#*UID: fplc_user<br> 
#*PW: 4020<br>   
#Start “Unicorn” program on computer<br> 
#*UID: Moshe<br> 
#*PW: 123456<br>   
#Select “System Control” tab on taskbar<br> 
#Under 'File' menu select “Run”<br> 
#Select the “Wash S100” program<br> 
#Click “Next” until window with “start” appears, click “Start” 
#Check column depositor to ensure buffer is running into the waste container.<br> 
#*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:<br> =====

#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<br> 
#*Flow rate should be 0.25ml/min<br> 
#*Load volume should be 1.2ml<br> 
#*Collection Volume should be 1.2ml/tube<br> 
#*Elution volume should be 1.5 column volumes (180ml)<br>   
#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 &amp; 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<br> 

===== 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.<br>