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Welcome to the Integrated Department of Immunology at the University of Colorado - School of Medicine and National Jewish Health.

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Running a K/M lab sizing column

Protocol for running a sizing column in the KM lab

Starting the instrument:
  • Turn on the computer and the FPLC.  Login (there is no password).
  • When you turn on the program, the fraction collector will move around so make sure that the fraction collector is not going to bump into the ejection valve (you can take off the fraction collector or adjust the valve to the correct height).
  • Open the FPLC program, login as Fran.  A window should pop-up saying “scanning for instruments”.
  • While the computer is connecting to the instrument, wash the pump lines with water (dip each line in the 50mL conical water tube) and transfer to the PBSA bottle.
  • Exchange the syringes attached to the instrument (should be EtOH syringes) for PBSA syringes.  Make sure your syringes do not have any air bubbles in them.
  • Wash the injection loop by pushing at least 3mL of PBSA through the bottom syringe (it should drip out the injection hole, catch in a waste container).
  • Wash the pumps with PBSA: on the computer, go to “manual,” “pump,”  “pump wash,” and click on both pumps A and B.  Hit “execute”.  This takes about 3-4 minutes and the instrument panel should read “washing”.
  • When the wash is done, start washing the sizing column with PBSA.  Go to “manual,” “pump.” Run PBSA through position 1-bypass at 5mL/min until the baseline stabilizes (~1 min). Hit “End”.  The instrument should beep.
  • Go to “manual,” “pump”, “column position” and switch the column position from “position 1-bypass” to “position 3”.  Hit execute. This is Belle, the sizing column for people outside of the KM lab (guest users).  Make sure that Belle is attached to column position 3 before starting.
  • Go to “manual,” “pump,” and run the column at 0.5mL/min through column position 3.  NEVER RUN FASTER THAN 0.5mL/min or you will collapse the column.  Wash with at least 10mL PBSA (20 minutes).  The baselines should be stabilized.
Beginning your run:
  • While the column is washing, concentrate your protein down to at least 500uL in a centricon. The smaller your injection volume, the better separation you will get, so its best if you can go down to 300uL.  The amicon ultra 15mL centricons (Cat No. UFC903024) are best for concentrating because they reduce aggregation on the filter (more surface area) and they are faster.  
  • Add 1/10th volume of 30% sucrose and filter through a 0.2uM filter (yellow cap).  THIS IS VERY IMPORTANT; IT WILL PREVENT YOU FROM CLOGGING UP THE COLUMN.  Once a column is clogged it has to be repacked, which is not a fun process.
  • Load the purple fraction collector with glass tubes through row H.
  • Adjust the ejection valve to the correct height, use the ruler as a guide.
  • Obtain an injection needle from the KM lab and put about 200uL PBSA in the syringe (no air bubbles).  Carefully load your sample, do not turn the syringe upside down and keep the protein (in sucrose) separate from the PBSA.  The PBSA allows you to inject the entire amount of protein into the instrument.
  • Turn off the column wash (hit “End”, the instrument should beep).  
  • Insert the syringe, making a liquid-to-liquid contact by pushing a little PBSA through the loop with the bottom syringe.  
  • Turn the valve by the injection site down to waste (perpendicular to the injection site, this will put the PBSA currently in the injection loop into the waste).  Slowly inject your sample.  Switch the valve back up (parallel to the injection site).
  • On the computer, go to “Run”.  Open the folder “Jill’s” (inside of Fran’s folder).  Select “BelleV310mm”.  This method will run the sizing column at 0.5ml/min on column position 3.  It uses the purple fraction collector with glass tubes.
Finishing the run and shutting down:
  • Your data will be saved in Rachel’s folder. You can open and print it in the evaluation window.
  • Wash the sizing column with PBSA.  Go to “manual” “pump” “column position” and switch the column position to position 3, hit “execute”.  Go to “manual” “pump” and run PBSA at 0.5mL/min for at least 20mL.
  • When the wash is complete, hit “End”.  The instrument should beep.  
  • Rinse the lines in water (dip each line the water 50mL conical tube) and transfer into the 20% EtOH bottle.  
  • Switch the syringes back to the EtOH syringes, and wash the injection loop by pushing at least 3mL of EtOH through the bottom syringe.
  • Wash the pumps in EtOH, “manual”, “pump”, “pump wash” and switch both pumps A and B on.  Hit “execute”.  
  • After the pump wash, close the FPLC program and shut down the computer.  Turn off the instrument.
Most proteins will need protease inhibitors shortly after the run, we usually add these to the fractions we want.  We also add azide to the final tetramer and concentrate it down to a reasonable volume (~1mL).