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发表于 2006-11-11 00:52:54 |只看该作者

Native Isoelectric Focusing Free-flow Electrophoresis (IEF-FFE) Fractionation of Crude Protein Mixtures
Peter J.A. Weber, Gerhard Weber and Christoph Eckerskorn
This protocol was adapted from "Native IEF-FFE Fractionation of Crude Protein Mixtures," contributed by Peter J.A. Weber, Gerhard Weber, and Christoph Eckerskorn, Chapter 19, in Purifying Proteins for Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2004.

INTRODUCTION

This protocol describes a method for fractionation of a native protein sample using a free-flow electrophoresis instrument. This protocol is used in situations where inter- or intramolecular protein interactions are important for the stability of the target protein (or protein complex), the activity of an enzyme, or the success of the separation in general. For fractionation under denaturing conditions see Denaturing Isoelectric Focusing—Free-Flow Electrophoresis (IEF-FFE) Fractionation of Crude Protein Mixtures.

MATERIALS

Reagents

Concentrated protein sample

H2SO4 (100 mM)

This is the electrolyte for the anode circuit. The minimum amount needed to run routine, short experiments is 400 g. Use larger amounts for longer-term experiments, i.e., more than 12 hours.

Isopropanol

Kerosene (low odor)

NaOH (100 mM)

This is the electrolyte for the cathode circuit. The minimum amount needed to run routine, short experiments is 400 g. Use larger amounts for long-term experiments, i.e., more than 12 hours.

Native anode stabilization media (Inlet I1)

Native cathode stabilization media (Inlet I7)

Native counterflow media (Inlet I8)

Native separation media 1 (Inlet I2)

Native separation media 2 (Inlet I3-I5)

Native separation media 3 (Inlet I6)

Pro Team reagent set (TECAN), including: ProLyte 1, ProLyte 2, ProLyte 3, HPMC, Glycerol, SPADNS (sulfanilic acid azochromotrop), and colored pI markers

The ProLyte reagents are the major ingredients of the separation media. ProLytes are precisely defined mixtures of low-molecular-weight organic acids, bases, and zwitterions (<300 Da). They form linear pH gradients from at least pH 3.0 to pH 11.5 and allow highly reproducible runs as well as easy removal from the proteins.

Equipment

Conductivity meter (10 µS/cm - 200 mS/cm) and a 96-well absorbance reader equipped with two filters (abs = 500-530 nm, abs = 400-420 nm)

Gloves (powder-free PVC or nitrile)

Microtiter plates (96-well, standard and deep-well)

Pro Team FFE instrument (TECAN) equipped with: 7 x 0.64-mm I.D. (orange-white) media tubes, 1 x 1.42-mm I.D. (yellow-yellow) counterflow tube, 1 x 0.51-mm I.D. (orange-yellow) sample tube, 0.4-mm spacer, 0.65-mm filter paper strips, and 96-well fraction collector

Water cooler (P350 W) and a pH meter equipped with a micro pH electrode (diameter of the probe = 3-5 mm)

METHOD

Disassembling and Cleaning the Instrument
1. Turn on the cooler and set the temperature to 10°C. The separation chamber must be in thermal equilibrium before it is opened.

2. Set the separation chamber to the vertical position and attach the fractionation plate via the magnetic holder. Open each pair of clamps on the separation chamber and carefully pull open the front.

3. Remove the electrode membranes and soak them in a 1:1 mixture of glycerol and isopropanol.

4. Remove the strips of filter paper that cover the electrodes and immerse them in H2O.

5. Connect the sample tube to the inlet closest to the cathode (S3) and close the other two sample inlets. Use a lint-free paper towel to clean the inside walls of the separation chamber. Clean first with H2O, then isopropanol, kerosene, isopropanol, and finally again with H2O.

Sample inlet is merely used for separation of basic proteins.

Use a new paper towel for each cleaning operation. Perform the cleaning process twice on new equipment and after repairs or service.

IMPORTANT:
Never allow silicone, oil, grease, or adhesive to come into contact with the inside of the separation chamber—this could affect separation performance. Never use acetone! Prolonged treatment with kerosene and isopropanol may damage seals or other parts of the separation chamber. Use only powder-free PVC or nitrile gloves. Do not use powdered or silicone gloves!

Assembling the Instrument
6. Pre-wet the 0.4-mm spacer with H2O and place it on the front plate of the separation chamber, making sure that it is positioned centrally with respect to the electrode seals and the internal separation chamber seal. Do not cover the separation media inlets.

7. Place membranes on the anode and cathode, respectively. Lay each membrane from the top to the bottom and make sure that the smooth side of the membrane is facing the electrode seal. Do not allow the membrane to protrude over the electrode seal.

IMPORTANT:
When reusing membranes, make sure that a strip that has been used on the anode is not later used on the cathode, or vice versa.

8. In a similar manner (i.e., from top to bottom), place filter paper strips on top of the anode and cathode membranes, respectively.

IMPORTANT:
When reusing paper strips, make sure that a strip that has been used on the anode is not later used on the cathode, or vice versa.

9. Loosen the separation chamber clamps (turn counterclockwise) and, holding the second connecting bar from the top with both hands, close the chamber. Make sure that the front plate is in line with the back plate.

10. Close the middle two clamps simultaneously, and then close the adjacent clamps, pair by pair. When all the clamps have been closed, tighten them pair by pair. Tightening the clamps should displace any water that has been trapped beneath the spacer. When the water has been displaced, the spacer will appear clear.

11. Check that the membranes and filter papers have not moved. If necessary, reopen the chamber and repeat the positioning process. Place the fractionation plate on the fraction collector housing.

Filling the Instrument with H2O
12. Tilt the separation chamber to 45° and turn the bubble trap to the filling position. Immerse all media and counterflow tubes in a fill bottle containing H2O.

Never fill the chamber directly with separation media. This would introduce bubbles, which are impossible to remove, into the chamber.

13. Place the sample inlet tube in an empty microfuge tube. Do not tighten the adjusting screw. Open the three-way tap on the counterflow tube in all directions and also open the Luer-lock closure on the tap. Place the upper part of the counterflow tube in the fractionation tray and the other part in the bottle grid.

14. Start the media pump (direction "IN"), then close the tube cassettes that fix the tubes inside the media pump, and continue pumping at 50 rpm until the separation chamber is half full.

15. Reverse the pumping direction and empty the separation chamber until the inlet areas of the spacer are empty and all air bubbles have been expelled. For this stage, it is advisable to reduce the pump speed to 20 rpm.

16. Reverse the media pump once more and, using a flow rate of 50 rpm, fill the chamber, without introducing any air bubbles, to a level just below the fractionation tubes. At this point, increase the pump speed to 99 rpm and continue pumping until the counterflow reservoir is almost full.

17. To fill the last part of the counterflow reservoir, reduce the pump speed to 20 rpm.

The reduced speed should prevent bubble formation in the chamber. If air bubbles do appear in the separation chamber, drain the chamber to the level of the lowest bubble and repeat Steps 12-17.

18. When the counterflow reservoir is completely full, increase the pump speed to maximum and fill the counterflow tube and the bubble trap. Then reduce the flow rate to 20 rpm and connect the Luer-lock closure of the three-way tap by holding both openings up.

Any air bubbles remaining in the valve or Luer adapter can be displaced by gentle tapping.

19. Close the outlet opening of the three-way tap. The fractionation tubes will start to drip.

20. Tilt the chamber to horizontal and tap the fractionation plate. Check whether all 96 fractionation tubes are flowing freely. Clear any obstructed tubes by inserting a syringe into the opening and applying negative pressure until flow is restored.

When restarting the media pump (direction "IN") use the highest flow rate for a few seconds. When all the fractionation tubes are dripping, return the pump to normal speed.

21. Dry the outside of the separation chamber and check for leaks. If a leak is detected, the chamber must be drained, opened, and closed again.

22. Start the sample pump at 4 rpm (direction "IN"). Tighten the adjusting screw on the sample inlet tube until it starts to deliver the sample. Tighten the adjusting screw an additional quarter turn.

23. Change the pumping direction to "OUT" and run until no air is left in the tube. Then stop the sample pump.

Checking the Flow Profile
24. Dilute 500 µl of the red dye SPADNS with 50 ml of H2O. Position the H2O-filled separation chamber horizontally and switch off the media pump.

25. Place the media tubes for inlets I2, I4, and I6 in the dye solution and leave the remaining tubes for inlets I1, I3, I5, and I7, along with the counterflow tube for inlet I8, in H2O.

Never put the dye solution in inlets I1 or I7—this would contaminate the electrodes.

26. Run the media pump at 40 rpm (direction "IN") for 6 minutes. Then collect the resulting fractions in a 96-well plate. When a flow profile has been obtained, switch off the media pump and return the media tubes for inlets I2, I4, and I6 to H2O. Run media pump at 40 rpm (direction "IN") and rinse for 6 minutes. If the instrument has been set up correctly, alternate red and colorless stripes, of identical width and with sharp boundaries, will flow in parallel along the separation chamber.

Calibration of the Sample Pump
27. To calibrate the sample pump, fill a microfuge tube (the calibration vessel) with approximately 1.5 ml of H2O. Accurately quantify the contents of the tube by weighing to the nearest milligram.

Calibrate the sample pump after each adjustment of the sample pump screw.

28. Immerse the sample tube in the calibration solution. On the Pro Team FFE touchscreen, call up the "Calibrate sample pump" dialogue and follow the on-screen instructions. Weigh the calibration vessel again and save the calibration according to the on-screen instructions.

Calibration of the Media Pump
29. To calibrate the media pump, fill an appropriate bottle with approximately 200 ml of calibration solution (i.e., H2O) and weigh to the nearest 10 mg.

Calibrate the media pump every week or after changing the sample tubes.

30. Immerse the media tubes (inlets I1-I7, without counterflow tube I8) in the calibration solution. On the Pro Team FFE touchscreen, call up the dialogue "Calibrate media pump" and follow the on-screen instructions.

31. Weigh the calibration vessel again and save the calibration according to the on-screen instructions.

Filling the Instrument with Media and Electrolytes
32. Make sure that the media pump is switched off and the separation chamber is in the horizontal position. Immerse the liquid circuit tubes in the appropriate media (positive: anode circuit, negative: cathode circuit).

33. Close the safety cover of the electrode circuit bottles and the media pump. Turn on the electrode pump and check that it is working properly (as indicated by flowing air bubbles in the electrode ducts).

34. Immerse the media tubes in bottles containing the appropriate solution, i.e., separation or counterflow medium. Turn on the media pump at 20 rpm for 3 seconds (direction "OUT").

35. Tap the media bottles against the bottle grid to remove any bubbles that might remain at the tip of the tubes, and reverse the media pump (to direction "IN"). Run at 20 rpm for 15 minutes.>

36. Rinse the chamber with media for 15 minutes. Then adjust the rate of delivery to approximately 57 ml/hour to allow a constant flow of separation and counterflow media through the chamber.

Until the instrument is calibrated, the flow rate from the pump can only be approximated. After calibration, particularly in the actual sample separation context, it is important to use exact ml/hour values. The value 57 ml/hour was found empirically to work best for this protocol.

Operation
37. Set the voltage to 1500 V and the current to 50 mA, and then switch on the high voltage.

The high voltage cannot be switched on if (a) the safety circuit is open, or (b) the electrode pump or (c) the media pump is switched off, or (d) a connected cooler is not turned on.

38. Wait approximately 15 minutes for the current to reach a stable minimum of approximately 22 ±3 mA (17 ±2 mA for Protocol 2).

Checking the Performance of the Instrument
39. Dilute the FFE pI marker 1:10 with native separation media 3. The pI marker is a mixture of seven dyes (one red and six yellow) of different pI values.

40. Keep the separation chamber horizontal and pump the pI markers into the cathode sample inlet (S3) at a rate of 0.5 ml/hour.

41. Watch for the appearance of the first colored drops at the fractionation outlets. Run the test for 10 minutes after the drops appear.

42. Evaluate the results of the test by collecting the separated pI marker fractions in a 96-well microtiter plate located in the collection drawer. Avoid cross-contamination during sample collection by tapping the fractionation plate on the fractionation collector housing just before introducing and removing the drawer. The width of the individual pI markers should be no more than three to four wells.

Preparation and Application of the Sample
43. For efficient separation, ensure that the chemical and physical properties of the sample and the separation media are similar, particularly with respect to density, conductivity, and viscosity. To achieve this, prepare a concentrated sample solution and dilute it at least 1:5 with separation medium.

As a starting point, make sure that the working concentration of the sample is approximately 1-5 mg/ml. If no precipitation occurs, increase the sample concentration for successive experiments.

Most samples are colorless, which makes it difficult to monitor the progress of the experiment. To solve this problem, add 1% (v/v) Pro Team SPADNS to the sample prior to loading.

The total salt concentration of the sample should be no more than 25-50 mM. If the salt content of the sample is too high, either desalt it (e.g., using a desalting column or spin column) or dilute it with separation media.

Protein samples can appear turbid if protein precipitation has occurred or if insoluble cell components are still present. Clarify turbid sample solutions by filtration or centrifugation before use.

It is possible to add nonionic detergents such as CHAPS, octylglucoside, and Triton X-114 (0.11%) and reducing agents such as dithiothreitol (up to 50 mM) or uncharged phosphines to the sample.

44. To avoid carryover when applying a new sample, make sure that the separation chamber and the sample tube are rinsed for at least 30 minutes (media pump direction "IN," 57 ml/hr; sample pump direction "OUT," 2 ml/hr) prior to sample loading.

45. To apply the sample, stop the sample pump and snip the end of the sample tube. Snipping the end of the sample tube creates a small bubble in the tube, which is visible as a sharp border between media and sample.

46. Then immerse the sample tube into the sample vial. Turn on the sample pump (direction "IN") at a high flow rate until the bubble reaches the sample inlet. Then apply the sample at a flow rate of approximately 1 ml/hour.

47. As soon as the red dye reaches the fractionation manifold, start collecting the sample fractions in a 96-well plate. Continue sample collection for approximately 5-10 minutes by placing the microtiter plate on the drawer and moving it under the fractionation tubing outlets.

Collect these early fractions separately, because it may take some time for the fractionation pattern to reach its equilibrium state. However, when using small quantities of sample, avoid separate pre-sample collection, despite the fact that this may lead to lower resolution fractionation.

Avoid cross-contamination during the collection by tapping the fractionation plate on the fractionation collector housing just before introducing and removing the drawer.

48. Replace the original collection plate with a deep-well plate and continue to collect the sample fractions until the red dye leaves the separation chamber. Check the sample volume and the media volume regularly during the separation run.

49. Do not allow bubbles to enter the separation chamber. If bubbles enter the chamber, interrupt the run and empty and refill the chamber.

Check regularly for precipitation of the sample in the separation chamber. Precipitation appears as white lines in the chamber and can be tolerated as long as no immobile "islands" are formed. If such islands are observed, slow the sample delivery rate or reduce the concentration of the sample.

50. Use a fresh 96-well plate to collect the remaining sample fractions until the red dye has completely passed through the apparatus.

End of Operation, Active Rinsing
51. At the end of a separation run, switch off the high voltage. Stop the media pump and the electrode pump and keep the separation chamber horizontal.

52. Place the counterflow tube and the media tubes in a bottle containing at least 1 liter of H2O. Place the sample tube into an empty microfuge tube.

53. Restart the media pump and run at 15 rpm for 10 minutes (direction "IN"). Then increase the media pump flow rate to 50 rpm (direction "IN") and continue to run for a further 30 minutes.

54. At the same time, rinse the sample tube by running the sample pump at 4 rpm (direction "OUT").

55. Rinse the electrodes as follows:

i. Remove the two "longer" electrode tubes labeled + or – from the electrode media.

ii. Run the electrode pump until the electrode ducts are almost free of electrode media.

iii. Switch off the electrode pump and remove the bottles containing electrode media.

iv. Place all four electrode tubes in a vessel containing approximately 500 ml of H2O.

v. Operate the electrode pump for approximately 10 minutes.

vi. Remove the two "longer" electrode tubes labeled + or – from the H2O.

vii. Operate the electrode pump until electrode ducts are almost free of H2O.

viii. Switch off the electrode pump. IMPORTANT: To avoid injuries, always wear gloves when handling electrode media.

Passive Rinsing
56. Exchange the 96-well plate drawer for a rinse tray containing approximately 2 liters of H2O and run the media pump at 20 rpm (direction "IN").

57. Immerse the fractionation plate into the rinse tray and stop the media pump.

58. Place the counterflow and media tubes on the bottle grid and remove the rinse tray containing H2O. Turn the bubble trap to the draining position and tilt the chamber 45° upward.

59. Open each pair of clamps and loosen by turning counterclockwise (three complete revolutions). Close the loosened clamps.

60. Open the upper and lower pairs of clamps completely.

61. Remove the media pump safety cover and release the tube cassettes by pushing on the lower right-hand side.

62. Release the adjusting screw on the sample pump and place the sample tube on the bottle grid.

63. Switch off the FFE and let it stand overnight.

64. If the instrument is to be used within the next 24 hours, it can be left in this state. Otherwise, open the chamber. Rinse the spacers and filter papers with H2O and store them dry. Store the membranes in glycerol:isopropanol (1:1). Switch off the cooler.

TROUBLESHOOTING

Problem: No alternate red and colorless stripes of identical width and with sharp boundaries flow in parallel along the separation chamber.

[Step 26]

Solution: The problems may be due to the following: (1) unevenly tightened clamps, (2) surface contamination of the separation chamber, (3) incorrectly positioned spacer, seals, paper strips, or membranes, (4) partially clogged or leaking media tubes, (5) opened sample inlet (i.e., incorrectly adjusted screw on sample pump), (6) defective tubes or tubes of different diameters. Each of these parameters should be examined before repeating this procedure.

Problem: The width of the individual pI markers is broader than three to four wells.

[Step 42]

Solution: Broader bands are indicative of incorrectly prepared separation media or incorrect assembly of the separation chamber.

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