Purification Using Gradifrac

 

NOTE: This protocol is written for TraI36 but much of the usage of the gradifrac is generally applicable!

 

Some things to keep in mind

  1. Everything in the computer is expressed in concentration of Buffer B
  2. Always keep the protein on ice
  3. Keep your protein out of the loop as long as you possibly can

 

Using the Gradifrac

  1. Sign up for use of the Gradifrac a few days in advance
  2. Prepare and Ice bath
  3. Take one of the 500ml cell pellets that you made from the -80 freezer and keep in the ice bath during down time.
  4. Resuspend the cell pellet in 50mL of the first buffer that you will be using during purification.  (Usually 50mM NaCl, 50mM Tris (pH 7.5), 1mM EDTA.)
  5. Add 50uL of 100mM PMSF in 200 Proof EtOH
  6. Pour off the entire solution into the metal sonicator cup and sonicate in the Fambrough Lab.
  7. Sonicate at max Watt four times with a 2 – 3 minute breaks in between.  Be sure to place the metal cup on ice during breaks.  * DO NOT LET THE TIP OF THE SONICATOR COME OUT OF THE LIQUID OR YOU WILL END UP WITH A LOT OF FOAM WHICH IS DENATURED PROTEIN.
  8. Add another 50uL of 100mM PMSF in 200 Proof EtOH
  9. Split the solution in the metal sonicator cup into two of the smaller plastic centrifuge tubes over by the sink.
  10. Spin out these two tubes in the large centrifuge in the Fambrough Lab at speed 10,000 for 10-12 minutes.  Collect the supernatant
  11. Keep the supernatant on ice until you are ready to load the sample into the Gradifrac.
  12. Turn the four switches on the Gradifrac on.
  13. Make sure that the hoses are in the bottle of dH2O
  14. Scroll down to <MANUAL RUN> on the machine and hit <ENTER>
  15. Enter concentration of <50%> for concentration of B and hit enter
  16. Enter a volume of <5ml/min> and run for at least 5 minutes so that all the hoses are washed with water.
  17. Rinse and reassemble to loop being careful not to allow any air bubbles into the loop.
  18. Remove the small 3ml syringes that are connected to the black lever section and replace the hoses from the loop.
  19. Hit <END> on the machine so that the water stops running through the machine.
  20. Rinse off the ends of the hoses that are in the bottle of dH2O and replace them into the buffer solution that you will use. 
  21. Scroll back down to <MANUAL RUN> and hit <ENTER>
  22. Enter a concentration of <0%> for the concentration of B and hit <ENTER>
  23. Enter a volume of <5.0ml/min> and run for at least 5 minutes so that the hoses are washed completely with Buffer A
  24. After 5 minutes have passed do not stop the flow of the buffer but instead scroll up to the flow rate and change it to <1.0ml/min> and hit <ENTER> (You change the rate back to 1.0ml because you will be loading the column which is filled with ethanol which creates a lot of back pressure)
  25. Disconnect the hose at the spot where the column will be loaded
  26. Unscrew the top and bottom of the column off (the top side has red) and begin to fill the column with the buffer in the same manner you do when storing the column.  Once the column is full with buffer put some extra drops on the top and screw the hose onto the top of the column making sure tom minimize the air bubbles.
  27. Once you see that there is a flow coming from the bottom of the column, begin to fill the hole that the column will go in with the buffer coming out of the column.  Once it is full carefully screw the bottom of the column into the machine while making sure there are no air bubbles. 
  28. Once the column is securely attached allow the flow to continue at 1.0ml/min for another 5 minutes to make sure all the ethanol is removed from the column.
  29. Once the five minutes has passed Scroll up to the concentration and make sure that the concentration is set to <0% B>
  30. Change the flow rate back to <5.0mL/min> and run this wash for 5 minutes.
  31. After 5 minutes scroll up to the concentration and change the concentration to <100% B> and allow this wash to go for another 5 minutes.
  32. While this wash is going on take the caps off of the pens and replace them on the recorder.
  33. Once that wash is done scroll back up and change the concentration back to <0% B> and allow it to run for at least 5 minutes.
  34. After a few minutes of this wash turn the recorder on
  35. Wait for the blue pen to show a nice straight baseline
  36. Once you have a nice baseline make sure the black lever is on the <LOAD> position
  37. Then using a 10ml syringe inject your sample into the loop.  If you are doing the Blue Column be sure to dilute your sample about 1:5 with the buffer you will be loading it with (Buffer A) before loading it into the loop.
  38. Once all your sample is loaded into the loop push the black lever to the <INJECT> position so that your sample will begin to enter the machine
  39. Make sure to collect the flow through just in case something goes wrong
  40. Once there is about 1ml of your sample left push the lever back to the <LOAD> position so that no air bubbles are injected into the system.
  41. As your sample enters the machine the blue pen will go all the way to the top and will read at the extreme end.  Once the blue pen begins to have a baseline again you can begin to collect fractions.
  42. Set the fraction volume at 1.5ml and press the <ENTER> button
  43. Then enter the percentage gradient (usually 45% for the Heparin column and 55% for the Blue column)
  44. Then enter the amount of volume you want the gradient to occur over à a good rule of thumb is to have it so the percent and the gradient are equal i.e. if you set the gradient to 45% set the volume you want the gradient to occur over at 45ml.
  45. Once you hit enter the fractions will begin to collect.  Keep an eye on the recorder and when the blue pen records something you should write on the graph what number fraction it is for later reference. 
  46. Once all the fractions have been collected the baseline should return.  Once the baseline has returned use a 5ml syringe to load 5ml of urea into the loop.  Then push the black lever to the <INJECT> position so that the urea enters the system.  Be sure to indicate on the graph when the urea entered into the system.
  47. The blue pen should make some more shots towards the top of the graph as more stuff comes off the column.
  48. Once there is about 1ml of the urea left push the black lever back to the <LOAD> position so that no air bubbles enter the system.

 

Storing a Column When Finished

 

  1. Unscrew the column from the machine being careful not to twist the hose only turn the black part that is not part of the hose
  2. Reattach the hose from the column to the Gradifrac
  3. Using the ethanol syringe located by the Gradifrac, fill the whole column with 20% ethanol.
  4. When the column becomes filled with ethanol put one more drop on the end and screw the syringe into the column ensuring that there are no air bubbles in the column.
  5. Carefully squeeze the ethanol through the syringe.
  6. Repeat the cleaning procedure with the ethanol two more times for a total of three washes.
  7. On the last cleaning when there is about 5ml of ethanol left in the syringe screw on the bottom of the column so that the ethanol will not be able to run through.  Be sure to continue to apply pressure while attaching the bottom so that there are no air bubbles inside the column.
  8. Then continue to add ethanol to the top of the column to make sure the column is completely full of ethanol. 
  9. Once the column is full of ethanol put a little extra on the top to ensure that there will be no air bubbles.  Then screw on the top of the column and place the column back in its box

 

Cleaning and Turning Off the Gradifrac

 

  1. Remove the hoses form the bottles of buffer and screw the tops onto the bottles of buffer
  2. Rinse off the ends of the hose and any parts of the hose that was touching the buffer with the squeeze bottle of dH2O
  3. Then place the hose in large beaker of dH2O and seal with parafilm
  4. Push the black lever to the wash position and allow the water to run through the whole machine.
  5. Take the pens off of the recorder and put the caps on them and replace the pens in the drawer below the Gradifrac.
  6. Unscrew the two connections to the loop
  7. Carefully unscrew the black ends on the loop and remove the plastic covering.
  8. Carefully remove the tight black caps from the glass tube using the spatula by the sink. 
  9. Carefully clean all the parts with alconox detergent and rinse extremely well with dH2O. 
  10. Take the syringe with an adaptor on it that connects to the hoses and rinse both the hoses that connect to the loop with lots of dH2O
  11. Leave the loop unassembled and place carefully by the rest of the machine.
  12. Take two small 3ml syringes with a different adaptor on it that will connect to the two spots near the lever that the loop connected to.  Push some water into the system and leave the syringes connected.
  13. Clean off the bench, the area under the lever, and all the surrounding area with dH2O so that there is no visible salt build up.
  14. Empty the waste beaker into the sink.
  15. Turn off all four machines
    1. Recorder = switch on left side
    2. Computer = switch on back
    3. Control Unit = switch on front face

d.    Pump = switch on back