Hydrophobic Interaction Chromatography

You'll use two approaches for investigating protein in the pGLO lab. First you'll lyse the cells and use HIC to concentrate the proteins. Later, you'll use the protein samples from HIC in an SDS-PAGE gel.

This page covers the method for hydrophobic interaction chromatography (HIC) as used in the pGLO lab. You'll use this method to partially purify bacterial proteins, including GFP.

In the last couple of labs, you transformed some E. coli cells with a plasmid containing a gene for green fluorescent protein (GFP), saw the glowing colonies caused by the expression of GFP, and saw that you could induce the expression of that gene by taking advantage of an arabinose-specific transcription factor (AraC) and promoter. Then you started new cultures of cells containing the plasmid. What you’ve done so far is similar to the first few steps a biotech company would use to produce a protein from a cloned gene. Now you can do the next steps: lyse the cells and recover the GFP protein.

The protein purification problem

Cells make a lot of proteins. If you want a specific protein from a cell culture, you need to separate that protein from all the others, and from all the other molecules in the cell.
You’ve already used one method for separating macromolecules: electrophoresis. Protein and DNA electrophoresis both act on the same principle: some molecules migrate faster through a gel than others. Protein electrophoresis is a very sensitive way of separating different proteins from one another, but it has some limitations. For one thing, the protein is in the gel when you finish. If you want to use that protein for something, you’d need a way to get it out of the gel. Also, you can’t load very much protein on a gel. And finally, the method you used, SDS-PAGE (polyacrylamide gel electrophoresis), uses detergent (SDS) to denature the proteins. This means that you’re not likely to recover functional protein.

Column chromatography

Column chromatography is an alternative to electrophoresis for separating proteins or other macromolecules. In principle, it’s similar to electrophoresis: you apply a sample containing a mix of proteins, and the different proteins get separated from each other because some travel faster than others. The key differences are what makes the proteins move, and what slows them down.

  • Electrophoresis: proteins move because they’re pulled by an electric field. They slow down because they don’t easily fit through the small pores of the gel. Bigger proteins move more slowly. The gel is thin and flat.
  • Column chromatography: proteins move through a column because they are carried by a moving solvent (the mobile phase). They slow down because some stick to the column more than others, depending on the chemical nature of the proteins and the column. The column is cylindrical and filled with some kind of matrix (the solid phase). Many kinds of column matrices are available, for separating different kinds of proteins.
    In some kinds of column chromatography, the solvent is pumped through the column at a constant rate. In this experiment, we will use “spin columns” that fit into microfuge tubes and in the microcentrifuge rotor. You can simply apply the solvent to the top of the column and force it to flow through the column matrix by centrifugal force.

Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography (HIC) is commonly used for separating proteins. The principle is simple: the protein sample is applied to a column filled with a hydrophobic matrix, and the proteins stick to the column matrix by hyrophobic interactions.

The HIC column is filled with tiny, porous plastic beads that are coated with short nonpolar hydrocarbon chains. A protein sample is dissolved in an aqueous, salty buffer and applied to the column. The proteins can either stick to the column through hydrophobic interactions or remain in the solvent through hydrophilic interactions. Proteins contain some hydrophilic regions and some hydrophobic regions, so they show some degree of affinity for both the solvent (the salty buffer) and the stationary phase (the HIC matrix).

The overall hydrophobicity of a protein depends on the polarity of the side chains of the amino acids making up the protein’s primary structure. Cytosolic proteins such as GFP are much more hydrophilic than they are hydrophobic. That’s why they are water-soluble. (As you may recall from lecture, proteins contain both hydrophobic and hydrophilic regions; the water solubility of a protein depends on the polarity of the side chains of the amino acids making up the protein’s primary structure.) Such proteins are highly solvated in water, meaning that they are tightly hydrogen bonded with surrounding water molecules. In order to make the protein stick to the hydrophobic matrix instead of being dissolved in the water, it is necessary to weaken the hydrogen bonding. In this experiment, you’ll accomplish that by dissolving the proteins in and ammonium sulfate buffer. Ammonium sulfate weakens the proteins’s interaction with water and allows the hydrophobic interactions with the column matrix to play a greater role.

In HIC, proteins are applied to the column in a high-salt buffer and eluted in a low-salt buffer. In some experiments, researchers gradually decrease the salt concentration of the solvent, causing different proteins to elute at different rates (least hydrophobic proteins first). In your experiment, you’ll start with high salt to make most proteins bind, then switch to no-salt to make almost all proteins elute. Used in this way, HIC concentrates proteins. You can apply a large volume of your initial sample, and then recover the proteins in a smaller volume. You should end up with a concentrated sample of bacterial proteins, including GFP.

Another advantage of HIC is that it generally does not completely denature proteins as they are purified. Therefore, GFP remains fluorescent throughout the purification procedure. Hence, you’ll be able to check whether the protein is sticking to the column by looking for green fluorescence in either the column itself or the eluent (the liquid that goes throught he column).

Lysing the cells

The first problem that you face in trying to purify pGLO is that the protein is inside the cells. To get it out, you have to lyse (split open) the cells. You can achieve this using a two-step method:

  • Lysozyme, as the name implies, is an enzyme that causes lysis of bacterial cells. This enzyme eats away at the cells’ protective cell wall, weakening them. The TE buffer not only maintains the proper pH for lysozyme activity, but is hypotonic to the cells causing them to swell by osmotic pressure.
  • Freezing finishes the job of lysing the cells. When ice crystals form, they break open the weakened cells.

Once you’ve lysed the cells, you’ll have a crude mixture of cell contents (a lysate). You can pellet insoluble cell debris by centrifugation, then apply the soluble lysate to the column.

Start the cultures

You'll start the liquid cultures one lab period, and use them the next.

As usual, you will want liquid cultures to produce the large numbers of cells to produce protein for this lab. You should prepare two liquid cultures, one with arabinose to induce GFP expression and one with no arabinose. The liquid culture media are usually supplied with 4 ml LB broth in culture tubes, ready to use. The arabinose is already added to some tubes.

You'll need:

  • Your pGLO plate. You may have several different plates with pGLO in the refrigerator. It's essential that you start with a plate that contains ampicillin, because that ensures that the cells contain the plasmid. Any plate with colonies containing pGLO wil work, but it's best if the plate doesn't contain arabinose.
  • One tube of LB liquid. Label your tubes with tape immediately, so you don't mix up the two different media. Label the tubes with tape; don't write directly on the tubes or lids.
  • One tube of LB liquid + arabinose.
  • Ampicillin stock solution. Check the concentration of this stock solution and add enough to make a final amp concentration of 50 μg/ml.

To inoculate the cultures, use a metal inoculating loop and transfer a tiny bit of a colony from plate with pGLO to each tube. Ideally, you would start with non-fluorescent colonies (from a pGLO plate with amp only or amp+arabinose+glucose). If you put a large amount of fluorescent cells into your no-arabinose culture, you'll have fluorescence where you're not supposed to have it. If you start with fluorescent colonies, make sure you use only a tiny amount of cells to start your cultures.

You'll inoculate the cultures one lab period and then let them incubate in the shaker incubator until the next lab period before continuing to the next section below.

Prepare lysates

You'll do this the following lab period, after letting the cultures grow.

In the last lab period, you should have prepared four liquid cultures of pGLO-transformed cells -- one culture with arabinose, one without. Now you need to prepare a lysate from each of these four. Perform all the "prepare lysate" steps below with all four samples, and then do the "HIC" steps with only two samples, as shown on the lab guide page.

Materials

  • Your tubes of liquid bacterial culture containing pGLO
  • 2 1.5-ml microfuge tubes for each sample (Use clear tubes, not colored.)
  • Micro tube rack
  • Lysozyme solution, on ice
  • 2 spin filters in micro tubes (for making HIC columns)
  • HIC resin (“Bio-Rad Macro-Prep® Methyl-HIC Support” macroporous polymeric beads)
  • Pipetmen, tips, beaker for waste tips
  • 1 empty glass culture tube for waste, along with a test tube rack to hold this tube

HIC buffers with specific concentrations of ammonium sulfate:

  • Equilibration buffer — A high-salt buffer (2 M (NH4)2SO4)
  • Binding buffer — A very high-salt buffer (4 M (NH4)2SO4)
  • Elution buffer — A no-salt buffer (10 mM Tris/EDTA; also called TE)
HIC 8083

Step 1: observe. Remove your liquid cultures from the shaker and observe them in normal room lighting and then with the UV light. Did they grow? (If so, the broth will be cloudy.) Are they fluorescent? Note the color of the fluorescence. A liquid culture without GFP may have some autofluorescence all by itself, but it’s white rather than green. Your + arabinose culture should be bright fluorescent green, with most of the fluorescence concentrated at the bottom, where the cells are. Record your observations; if you end up with unexpected results, you might want to be able to go back and review what you saw.

Culture tube

Step 2: transfer. You should have approximately 4 ml of each liquid culture. From each culture, pipet 2.0 ml into a 2.0-ml micro tube. Use the stuff on the bottom of the tube, because that’s where all the cells are. You should have four tubes.

Pipetting the culture into micro tube

 

Placing micro tubes in centrifuge.

Step 3: spin. Centrifuge for 5 minutes at high speed. Be sure to balance the centrifuge.

Fluorescent pellet; non-fluorescent supernatant.

After the spin, you should see solid bacterial pellets in all your tubes. At this point, the cells contain the protein you want. Observe the tubes under UV light and Record your results. Some of your pellets should be strongly fluorescent, and some not.

Remove supernatant.

Step 4: Pipet off the liquid supernatants into an empty glass culture tube for disposal.

Step 5: resuspend. Add 250 µl of TE buffer to each bacterial pellet. Resuspend the bacterial pellets thoroughly by rapidly pipetting up and down.

Step 6: lysozyme. Add 50 µl of lysozyme to each resuspended bacterial pellet and vortex or flick the tube to mix. Let sit at room temperature for 10 minutes. The lysozyme will start digesting the bacterial cell wall. The cells are also experiencing osmotic stress, since they are in a buffer with no salt; this helps lyse the weakened cells. Vortex for 30 seconds to speed the process.

Step 7: freeze. Put the tubes in the –80° freezer until they’re frozen solid (about 10 minutes). Freezing will complete the lysis of the bacteria. It’s essential that your samples freeze completely. Remove your tubes from the freezer and thaw them in your hand. Vortex to mix well.

Fluorescent supernatant.

Step 8: spin. Spin the tubes in the centrifuge for 10 minutes at high speed. This will pellet the insoluble bacterial debris. While you’re waiting for the 10-minute spin, you can start preparing your HIC columns (see below for instructions).

After the 10 minute centrifugation, remove your lysate tubes from the centrifuge and examine the tubes under the UV light. The insoluble bacterial debris should be visible as a pellet at the bottom of the tube. The supernatant contains all the soluble materials from the lysed cells – including GFP, other proteins, and many other kinds of molecules. For the +arabinose tubes, most of the fluorescence should be in the supernatant at this point, because the cells have been lysed. Check for fluorescence in the pellet and supernatant, and record your results.

If you see most of the fluorescence in the pellets, the cells did not get fully lysed. You should put the tubes back in the freezer and freeze and thaw them one more time to complete lysis. You might also need to add more lysozyme and incubate again. Don't go on to the next step until you are sure you have good lysis. Otherwise, you won't be able to get much protein for the next steps.

The liquid supernatants are your lysates. You should still have four tubes.


Step 9: Transfer the supernatants to new 1.5 ml tubes. Be careful to avoid the pellets. If your pellet is loose, you may have trouble with this step. If your pellet is a gelatinous blob that won't stick to the tube, you may find it easier to use your pipetter to suck the pellet out of the tube rather than pipetting the liquid away from the pellet. Each lysate should be about 275 µl. Throw away the tubes with the pellets and keep the liquids.

Step 10: Two of your lysates (green and blue, tubes 1 and 3) will be used for HIC. For these two samples, save 50 µl of each lysate in a separate tube (use a 500 μl micro tube). This little bit of lysate won't be used in HIC and will be used for electrophoresis in the next lab, along with your other samples. The other two lysates (tubes 2 and 4, which aren't expected to be fluorescent) should be saved for protein electophoresis.

Hydrophobic Interaction Chromatography

In the previous steps, you started with intact cells and used lysozyme and freezing to lyse the cells. After pelleting the insoluble debris, you now have lysates. The lysates contain all the cytosolic proteins, along with a range of other molecules (RNAs, nucleotides, sugars, amino acids, etc.). In the following steps, you'll force the proteins in the lysate to bind to a column under high salt conditions, wash away those other molecules, and then recover the bound proteins from the column under low-salt conditions.

The following steps are simple, but easy to mess up; this video shows how to do the steps correctly.

Step 11: Prepare the lysates to go on the columns. The lysates must have a high concentration of ammonium sulfate in order to stick to the HIC column. Add an equal volume of binding buffer to the tube containing the pGLO Green lysate. (The lysate should be around 275 µl. The volume of binding buffer must be approximately equal to the volume of lysate; by mixing your no-salt lysate with an equal volume of 4 M ammonium sulfate binding buffer, you’re making your lysate 2 M ammonium sulfate.) Do the same with the pGLO Blue lysate. These lysates are now ready to go on the HIC column; each lysate should have a volume of approximately 400 to 600 µl. Make the volumes the same to balance the centrifuge.

Micro tubes with column inserts.

Step 12: Prepare the columns: You'll need two columns, one for the green sample, and one for the blue sample. The columns start out as filters that fit inside a microcentrifuge tube. You'll need to prepare them by adding the HIC matrix. First, shake the bottle of HIC matrix thoroughly until all the microscopic white plastic beads are suspended in the buffer. Pipet 0.25 ml of HIC resin suspension into each spin column. Be careful to fully suspend the resin before pipeting. The beads sink rapidly, and if you’re not careful, you ‘ll end up pipetting only the liquid and not the beads. Label the columns with your initials and blue or green.

Spin the columns (inside their centrifuge tubes ) for about 30 seconds or low to pull all the liquid through the column. Make sure it all goes through; if it doesn't, you'll need to spin longer.

Equilibrate the columns by adding 0.5 ml of equilibration buffer to the top of the column. With the columns in their micro tubes, spin briefly (30 seconds or so on low) to pull all the liquid through the column. You should see a bed of chromatography resin about 4 mm high in each column. Pipet off the liquid from the lower tube. The column is now equilibrated with 2 M ammonium sulfate.

Fluorescent spin column in micro tube.

Step 13: Bind protein to column. You have already added binding buffer to your samples. Make sure you've removed all the liquid from the lower tube. Now add the entire volume of sample+binding buffer to the column. The column only holds about 500 µl, so if you have more than that, you'll have to do it in two batches. Load 500 µl of the green lysate (with binding buffer added to make 2 M ammonium sulfate, from step 11) onto the top of the HIC resin in one of the columns. Put the blue lysate in the other column. Spin for 30 seconds on low. The entire volume of liquid should pass through the column into the tube. Examine the column under UV light and record your results. The fluorescence should be in the column; pipet off the waste liquid from the lower tube.

If you have leftover, remove the liquid from the lower tube and then pipet the remaining lysate onto the columns. Spin for 30 seconds on low and examine the tubes and the liquid. Your columns should be brightly fluorescent at this point; check for fluorescence and record your results. Pipet off the waste liquid.

Step 14: wash column. Add 250 µl of equilibration buffer to the column and spin for 30 seconds on low. The entire volume of liquid should pass through the column into the tube. You used equilibration buffer to equilibrate the column before applying the sample to the column; now you're using the same buffer to wash various unbound molecules off the column, while leaving the protein bound. Make sure all the buffer goes through the column into the tube. The fluorescence should remain on the column at this point. Examine the column using the UV light and record your results.

Fluorescent eluent in bottom of tube; non-fluorescent column.

Step 15: elute. Transfer the column to a new, labeled tube. Add 200 µl of elution buffer (TE buffer) to the column and spin for 30 seconds on low. The lid of the tube might not close; it's ok to spin it at low speed with the lid open. The entire volume of liquid should pass through the column into the tube. Again, examine the column using the UV light and record your results. The fluorescence should be in the liquid now, not on the column. If most of the fluorescence is still on the column, you may need to repeat this step.

Your proteins should be in the last tubes; label these carefully and save them in the freezer (along with your previously saved lysates )for the protein gel in the next lab. These tubes will be called your HIC samples. Throw away the columns.

Before you leave

You should have:

  • 4 lysates in the freezer. Lysates 1 and 3 are the 50 µl that you saved before HIC, while lysates 2 and 4 are the entire volume, around 300 µl.
  • 2 HIC protein samples (Sample 1: pGLO Green and sample 2: pGLO Blue); approximately 200 µl each) in the freezer
  • Your re-streaked backup pGLO plate from last lab moved from the incubator to the refrigerator, if you did that last time.

Waste:

  • Gloves, paper towels, etc. go in the regular trash (not the biohazard, unless they’re contaminated).
  • Used spin columns, microfuge tubes and pipet tips go in the biohazard trash.
  • Glass culture tubes: take off the tape and put them in a rack in the dirty-glassware tub on the cart. Combine all the tubes into one rack if possible.
  • Beakers used for waste tips: dump the tips into the biohazard trash and put the beaker into the dirty-glassware tub on the cart (if there’s not a tub, check with the instructor).

Review

  1. Why did you do the first spin? Why did you do the second spin?
  2. What did the lysozyme do?
  3. Why did you put the tube in the freezer?
  4. How do you know if lysis occurred? What should you do if lysis did not occur?
  5. Which HIC buffers are more salty, and why? What would happen if you used the wrong buffer? Why is the binding buffer 4M, while the equilibration buffer is only 2M?
  6. What does “elute” mean?
  7. Why were you supposed to save a little of each lysate in a separate tube before you added the binding buffer?
  8. Did you end up with GFP? How do you know?
  9. Are there other proteins still mixed in with your GFP? How can you find out for sure?
  10. If you wanted to purify a specific protein so you could do further experiments with it, what would be the advantages and disadvantages of using HIC vs. SDS-PAGE? Why might you use both of these techniques together?
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