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Making MHC (Ld) teramers

Tetramer Protocol


 
Infect Hi5’s with virus according to the protocol.  Grow 6 days at 19oC.  
 
Harvest supernatants and filter into 1L bottles.  Add 10mL of 10% NaAz. Store in cold room until column is run.
 
Affinity 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 (without bubbles) to the bottom of the column.
  • Place the 1mL pipette in 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 contact.
  • Turn pump off and transfer 1mL pipette into the 5L beaker containing supernatants. Remove syringe from the bottom of the column.  Cover both flow through and supes beakers with foil.
  • Set the flow rate of the pump to 2-3.5 ml/min.  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.
  • Run overnight in cold room.
  • Make sure the column does not run dry.
  • Wash with approximately 100 mL PBSA.
  • Reattach syringe when finished.
 
What if column runs dry?
  • Take off the top of the column and add PBSA.
  • Use large pipette to mix.
  • Allow the column to repack, add PBSA carefully as needed.
  • Pump PBSA through tubing connected to the top of the column and reassemble without air bubbles.
  • Reattach syringe to the bottom.
 
Eluting Affinity Column on FPLC
  • Attach affinity column to the stand.
  • Dip the FPLC lines in the water tube to rinse and transfer them to appropriate buffers (line A into Buffer A and line B into Buffer B).  Turn on the FPLC and the computer.
  • There is no password to log on.
  • Open the program and sign in under Jen.  A screen will pop up saying the machine is ready.  Click Clear All button.  Under methods, select pumps and start a pump wash (switch both to on and hit execute). Load labeled tubes into tray.
  • When pump wash is finished, under Column position, switch pump to position 8 since this is where you usually attach the column to (this could change depending on what positions are open; under Flow, switch gradient to 20% B; under Flow rate, switch rate to 0.5 ml/min.
  • Hit execute and wait for liquid to drip out of position 8.  Push on syringe attached to the column until it drips and attach the tubing on the top of the column to the FPLC at position 8 on Valve 2, being sure to make a liquid-liquid contact. Remove syringe.
  • Wait for the bottom tube to drip and attach it to position 8 on Valve 3.
  • At this point, I would check just to make sure the protocol you are running runs at the same position at which you attached the column, but as of now the protocol 28.14.8S runs off Position 8 on the AKTA Prime FPLC machine in the K/M lab.
  • On the program, go to Run and select 28.14.8 affinity, hit next several times.
  • Name your file with the tetramer name and date.
  • Make sure your tubes are lined up with the fraction collector.
  • Hit start.
  • At about 45 minutes into the protocol, when the program goes to 50% B, the lines need to be changed.  Pause the program.  Switch both lines into PBSA and wash the pumps as before.  Then start again.  This step washes the affinity column and is necessary for the preservation of the column.
  • MHC-I proteins should come off at fractions #40-50 plus or minus a few fractions.
 
Biotinylation of the protein
  • Immediately add 600ul of neutralization buffer and 2.6ul PMSF and 26ul of leu/pep (in the –80oC) to each fraction.
  • Combine fractions into centricon tubes (2-4) and spin them down at 5,000XG in the fixed angle rotor (20 minutes/spin) until there is a total of about 1.0mLs.
  • Buffer exchange your sample by adding 10mM Tris (pH 8) to fill tube and spin again until there is about 1mL total volume.  Repeat if necessary.
  • Remove sample into 15mL conical tube, leaving a small amount in the centricon tube.  Vortex the tube and turn it upside down into a collection tube.  Quick-spin down.  Remove sample into the same 15mL conical tube.
  • Add about 100uL 10mM Tris to each centricon to rinse, vortex, spin, and remove sample into the same tube.  Repeat if necessary.
  • OD the sample.  (OD x dilution)/ 1.4 = mg/mL
  • Dilute your sample to 1.8 mg/mL and determine your total volume.
  • Divide (total volume)/8.  Add this many uL of Biomix buffers A and B. (3.63mL/8 = ~450uL Buffers A and B).
  • (20u / 3000 ug/mL) x total volume (after adding A and B) = uL of BirA enzyme to add (in –80oC).
  • Add more PMSF and leu/pep.  If your total volume is 4.5 mL, add 4.5 uL PMSF and 45 uL leu/pep.
  • Incubate at room temperature overnight.
 
 
Preparing sample for FPLC
  • Immediately the next morning, concentrate biotinylated protein over centricon columns as before until there is a total volume of about 200uL.
  • Transfer the sample to a filter eppendorf column and rinse the centricon.  Transfer remaining sample.
  • Add 1% IAA (if you have 200uL add 2.0uL IAA).  Incubate at RT for 30min.
  • Add 20uL of 10XPBSA and 20uL of 10X 60% sucrose (if you have 200ul).
  • Spin through column at 13,000rpm for 6 min or until all has gone through.
 
Size Exclusion Column (Pluto)
  • Obtain 0.7 needle for injecting sample from Rachel.  Load sterile syringe with ~100uL of PBSA, followed by the sample.  Make sure
  • Push syringe (PBSA) attached to the injector site until it drips; barely push on the sample syringe