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Making soluble TCR (CT-TCR)

TCR protien purification protocol (CTTCR and 2CTCR)


 

08/18/2004

Day 1:  Put supernatants over H57 antibody column
  • Obtain column tubing from Rachel in KM lab, pump from Jen’s bottom drawer, 1mL pipette (remove filter), and 2 5L plastic beakers
  • Place tubing in pump, attach one end of tubing inside the 1mL pipette so that it is flush with the bottom of pipette (attach with tape at the top).
  • Attach a syringe filled with PBSA and turn the pump on (make sure it flows in the correct direction).
  • Push on syringe and attach the tubing to the top of the column with a liquid-liquid connection.  Remove syringe from bottom of column and let it run to make sure it looks okay.
  • Turn pump off and transfer 1mL pipette into the 5L beaker containing supernatants.  Turn pump back on.  Cover both flow through and supes beakers with foil.
  • Set the flow rate of the pump.  This depends on how many liters of protein you have.  You can test it by starting it and determining how much goes through every hour then turn the speed up or down depending on that.  Keep in mind that it tends to speed up slightly with time.  Also, calculate so that enough is left over when you get back the next morning it is not dry, but almost done.
  • Run overnight in cold room.
  • Make sure the column does not go dry!
 
Day 2:  Elution of H57 column
  • Wash with approximately 100 mL PBSA.
  • Reattach syringe when finished.
  • I usually do the elution at the lab bench since it requires constant switching of the buffers and there is no method on the AKTA machine in the KM lab, only on the older machine.  It may still be on the old machine, but maybe not, because it broke after the method was written.  It is also just easier on the bench, I think.
  • Be sure before starting the elution that all the buffers are made:
PBSA= PBS with 5mM NaN3
20% B= 80 mL 50mM NaHCO3 =A; and 20 mL 50mM Na2CO3=B
50mM DEA=0.54 mL diethylamine in 100mL ddH20 pH is 11.8 unadjusted
50mM NaCitrate pH 3
1M Tris pH 6.9
  • Prepare about 70 tubes and you will want to collect 2mL fractions.  I just use the pump and transfer the tubing into the 20% B solution.  I then collect approximate 2 mL fractions for the first 25 tubes.  Then, turn the pump off, transfer the tubing into 50mM DEA, and then collect another 25 tubes.  Turn the pump off, transfer tubing into PBSA, and then collect another 12.5 tubes.  Run another 25 mL PBSA through the column, but there is no need to collect these fractions.  Turn the pump off, transfer tubing into 50mM NaCitrate, and put 50 mL through the column.  No need to collect these at all.  The NaCitrate just strips the column of any remaining junk. Then, transfer the tubing into PBSA again.  Wash 100mL or more through the column and then check the pH of the flow through coming out of the tubing.  You want it to be back up around pH7.  It is very important to wash the column after using the NaCitrate, as it would destroy the column if it were left at such a low pH.
  • Your samples should come off in the 50mM DEA fractions.  I O.D. those 25 tubes and collect the peak.  You will also want to be sure to add 600ul 1M Tris pH6.9 to neutralize your protein.  Do that as soon as possible.  Also, add protease inhibitors leupeptin/pepstatin(100X) and PMSF(1000X) to your peak samples.  
  • Concentrate the peak down to between 200ul and 2mL and keep it in a tube with protease inhibitors overnight.  (If you can concentrate down to 200ul that is optimal for running over the sizing column, however, if you have tons of protein, you can leave it as high as 2mL since that is the volume of the loop you inject into.)
 
Day 3: Run protein over the sizing column.
  • I would first O.D. your sample before running it over the sizing column.  If you have more than 5mg of protein, you may want to ask Fran about running it over the giant column in their lab.  The problem with this is that only Fran and Rachel can use that column.  If you do not feel comfortable bothering them, then you always have the option of running it over the smaller column twice.  
  • The smaller column is the same one we use to run the tetramer sizing columns.  It is called Pluto and you can use method name JWKplutovalve2 under Fran.  Last time I did this, my protein came off between Fractions 51-60.
  • Collect the appropriate fractions and add protease inhibitors.  Take an O.D. and record on the tube and in a lab notebook.  Keep in the refrigerator until you are ready to run a protein gel.
Day 4(or sometime soon after purifying your TCR):Test your protein on a gel and by flow cytometry
  • After running a protein gel to see how good it looks, you can test it on flow and see if it stains.  
 
Now you are done!