Chapter 4:

Section 4.3
Dialysis: Desalting and Buffer Exchange

Background

At this point your target protein, ß-galactosidase, is in a solution with a high concentration of salt, ammonium sulfate. The excess salt must be removed because it will interfere with the process of ion exchange chromatography, the next purification step. In addition, a different buffer is needed for the ion exchange chromatography step. There are several methods that can be used for desalting and/or buffer exchange. We will use dialysis, because it is relatively simple to set up and our sample size is small.

Dialysis tubing is a semi-permeable membrane available in a wide range of size dimensions and pore sizes (molecular weight cut-offs). Our procedure uses dialysis tubing with a 10,000 Dalton molecular weight cut-off. Our extract is placed inside the tubing and the ends are sealed off. The tubing is then suspended in a large volume of the desired buffer solution. The pores in the membrane allow molecules that are smaller than the pores to move freely across the membrane. Therefore, the ammonium sulfate ions will cross out of the tubing into the buffer. Eventually, an equilibrium is achieved where the concentration of ammonium sulfate is equal inside and outside of the tubing. However since the volume outside the tubing is much greater than inside, and this outside volume is replaced with fresh buffer, over time, most of the salt will leave the tubing (Figure 4.2). Larger molecules (such as most proteins) are retained within the membrane. Since most buffer components are small molecules and can therefore pass through the pores, dialysis is also used as a method for changing buffers.

Figure 4.2

Other methods for desalting and buffer exchange: Gel filtration chromatography is another method of desalting because it separates molecules on the basis of size. Smaller molecules can move inside the gel beads and therefore progress more slowly through the column than larger molecules, such as proteins, which remain outside of the gel beads. If the column has been equilibrated with the new buffer, the protein will elute in the new buffer, leaving the salts from the old buffer in the column. Tangential flow filtration is a high capacity method that is similar, in principle, to dialysis but large volumes can be applied to the same membrane system since the volume containing the target protein is recirculated through the same membrane system using a pump.

Procedure 4.3 Dialysis

Procedure 4.3a: Slide-A-Lyzer™ Dialysis (Pierce)

1. Read over the protocol in the Pierce product literature before you start. Locate materials, including large beaker with buffer, syringe (10-20 mls) with 18-Gauge, 1 inch beveled needle. Hold the cassette by the plastic frame only – do not touch membrane. You will need to locate the ports that are located at each corner of the cassette. Mark the ports as you use them and do not use them more than once. Do not allow needle to touch membrane. CAUTION: The beveled portion of the needle should penetrate the gaskets to only a slight degree – avoid overextending the needle into the cavity as it may puncture the membrane.
2. Hydrate membrane by placing cassette in 0.2M NTM buffer according to directions.
3. Fill syringe with sample, leaving a small amount of air in the syringe. (As noted in the product literature, maximum sample size for (NH4)2SO4 samples is 80% of maximum or 12 mls.)
4. With the bevel sideways, insert the tip of the needle through one of the syringe ports located at a top corner of the cassette.
5. Inject sample slowly. With the syringe needle still inserted in the cavity, draw up on the syringe piston to remove air from the cassette cavity. This will compress the membrane so that the sample contacts the greatest window surface area.
6. Remove the syringe needle from the cassette while retaining air in the syringe. Label the port that you have used and do not use this port again.
7. Attach the cassette to the buoy and place unit in beaker with buffer and stir bar.
8. Place the beaker or flask on a stir plate and stir at least two hours (overnight is OK) in the cold.
9. Change the buffer after allowing at least another two hours of stirring. Repeat this process two more times for a total of three changes over at least an 24 hour period. Ideally, you should not take longer than 2 days to accomplish three changes.
   
   To remove sample after dialysis is complete:
10. Fill the syringe with a volume of air equal to the sample size and, with the bevel sideways, insert the tip of the needle through a different syringe port located at a top corner of the cassette.
11. Inject air slowly into the cassette to separate the membranes.
12. Turn the unit so that the needle is on the bottom and allow the sample to collect near the port. Withdraw sample into the syringe.
13. Centrifuge the dialysate at 10,000 xg for 10 minutes to remove any insoluble material. Carefully separate the supernatant from the pellet.
14. Record the volume of the supernatant and remove 0.2-0.4 ml in 0.1 ml aliquots for assay. The supernatant that you will use for further purification may be stored in the refrigerator.

Procedure 4.3b: Dialysis Tubing:

1. Read over the protocol completely before you start.
2. Figure out how much dialysis tubing you will need to hold your sample and cut a piece that has an extra couple of inches. Follow manufacturers directions. Always handle dialysis tubing with gloves.
3. Wash your dialysis tubing. Place the tubing in a beaker with water. Place the beaker in the cold and stir for at least 2 hours. This will remove impurities, such as antifungal and antibacterial agents with which the tubing has been treated. Never let your dialysis tubing dry out once it has been wetted.
4. When washing is completed, remove the water carefully from the tubing. Clip the bottom of the tubing with a dialysis clip. Carefully pipette your sample into the dialysis tubing, being careful not to puncture the tubing. Also, don't spill your sample! Clip the top and attach a string to the top of the tubing.
5. Place the tubing in a beaker or flask containing approximately 1000 ml of 0.2 M NTM buffer and tape the string to the side of the container. Did you remember to add DTT to the NTM? (You may share beakers with other groups.)
6. Place the beaker or flask on a stir plate and stir at least two hours (overnight is OK) in the cold.
7. Change the buffer after allowing at least another two hours of stirring. Repeat this process two more times for a total of three changes over at least an 24 hour period. Ideally, you should not take longer than 2 days to accomplish three changes. Note:
8. Carefully remove the dialysate from the tubing with a pipette and squeeze out as much as possible of whatever is left into a centrifuge tube.
9. Centrifuge the dialysate at 10,000 xg for 10 minutes to remove any insoluble material. Carefully separate the supernatant from the pellet.
10. Record the volume of the supernatant and remove 0.2-0.4 ml for assay. The supernatant that you will use for further purification may be stored in the refrigerator.

Assays

• Determine the enzyme activity of the dialysate using the method outlined in Chapter 3 (Procedure 3.2).
• As time permits, determine the protein concentration using the Bio-Rad assay and calculate specific activity according to the assay methods in Chapter 3 (Procedure 3.1).
• Summarize the results of the purification so far by making a table as shown in Table 4.2. Include the data from each of the purification stages listed below.
  
  
  • The crude extract
  • The ammonium sulfate supernatant
  • The redissolved ammonium sulfate pellet
  • The dialysate

What do you think should be happening to specific activity as you go from crude extract to dialysate?

Assignment: Turn in a progress report of your purification procedure according to your instructor’s directions.

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Lisa Seidman
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(608) 246-6204

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