Protein Precipitation

Background
In the Gel Filtration lesson, we saw that immunologists can separate proteins from a mix by using a technique called gel filtration. In gel filtration, you will recall, a protein serum is added to a column of gel beads. The higher molecular weight proteins pass around the beads and quickly exit the bottom of the column. The smaller molecules pass into the beads taking more time to reach the bottom. Since the small and large proteins reach the bottom of the tube at different times, they can be collected in different containers.

Sometimes one technique for purifying a protein is not enough. To isolate an antibody, immunologists will sometimes do two separating procedures back to back. In this lab you will use one of the other protein separating techniques, protein precipitation. We use the word precipitation here in the same way as when we talk about rain; just as rain falls from the sky, we can make protein fall from a suspension.

As in gel filtration, molecular weight affects the precipitation of protein from a water suspension. But in protein precipitation, polarity also plays a role. The simplest forms of organic molecules are carbon chains with hydrogens attached. Such molecules do not mix with water because they have no polar groups. As you recall, nitrogen, oxygen, and fluorine are the most highly electronegative elements. They are the atoms that form hydrogen bonds. When they are present and exposed on the protein molecules, they will form hydrogen bonds with the water molecules. The hydrogen bonds allow the proteins to disperse through the water and remain suspended by it. To a lesser degree, other electronegative atoms and ions bond with the water.

If something could be added to a protein solution that would be strong enough to pull the water molecules away from the polar units on the proteins, then the proteins would no longer stay suspended. In a sense, the water molecules would drop the proteins. The proteins would fall to the bottom of the container.

Here is an analogy for protein precipitation: Imagine a party for two-year-olds. The celebration begins with Mom passing out apples to all the kids. For a couple of minutes, the children are happy munching the fruit. Then Dad walks into the room and panics. He is supposed to make an apple pie for the company picnic today! He leaves the room and comes back in half a minute carrying a bag. He dumps the contents onto the floor. The kids turn to see a pile of candy bars sitting there. Immediately, they drop their apples and grab the candy. Dad walks around and picks the apples off the floor.

In this lab you will separate a solution of proteins using protein precipitation. Since you will be testing the same protein mix that you used in the Gel Filtration lesson, you will pass the protein through a gel filtration column to identify which protein(s) precipitates. As a check, you will also test which protein remains in the supernatant, that liquid part of the mix from which the protein precipitated. You will need the procedure and the calibration curve from the Gel Filtration lesson.

Procedure - Day One

Add a 1.0 ml sample of the protein mix to a 100 ml beaker.
Drop a spin magnet into the beaker and place it on a stir plate at low speed.
Add 4.0 ml of saturated ammonium sulfate solution a drop at a time to the beaker.
Let the solution spin for ten minutes. Record your observations.
Transfer the entire contents of the beaker to a centrifuge tube. Make sure that you balance the centrifuge with a symmetrical arrangement of similarly filled tubes. See the teacher for help if you have any question about centrifuge balancing.
Let the proteins spin for one hour in the centrifuge.
Decant the supernatant into a collection beaker.
Add the supernatant to the beaker labeled "Class Supernatant Collection for Dialysis." MAKE SURE YOU ADD THE SUPERNATANT AND NOT THE RESUSPENDED PRECIPITATE! CHECK WITH EVERYONE IN YOUR GROUP THAT YOU ARE DOING THE RIGHT THING.
Add 1.0 ml of buffer solution to the centrifuge tube where the precipitate pellet is. Tap the bottom of the tube until the pellet is resuspended. Transfer the resuspended precipitate to the beaker labeled "Class Precipitate Collection for Dialysis." MAKE SURE YOU ADD THE RESUSPENDED PRECIPITATE AND NOT THE SUPERNATANT!
Observe the dialysis technique so you can answer Processing the Data question number one.

Procedure - Day Two

Set up the gel filtration column and collection tubes as in the Gel Filtration lesson.
Obtain a 10 µl sample of precipitate and separate it through the gel filtration column following the procedure given in the Gel Filtration lesson.
Do the Bradford assay as in the Gel Filtration lesson to identify the protein(s) appearing in the precipitate. Use the calibration curve to identify the molecular weight(s). Record your answers.
Dump the collection tube contents into the sink. Wash the tubes with multiple rinses of tap water and a final rinse with distilled. Flick out as much water as possible for the next collection.
Repeat steps 11 and 12 for the supernatant.
Dump the collection tubes and wash with soap. Rinse with distilled water.

Processing the Data

Why was it necessary to dialyze the precipitate and supernatant before testing them for proteins?
Below you will find a sketch of a portion of a protein molecule. Use it to make a drawing showing the hydrogen bonding relationship of water molecules with this section of the protein. In this drawing, assume the ammonium sulfate has not yet been added to the water. Next to this drawing, make another showing what happens to the molecules when ammonium sulfate is present. Label your drawings. <missing drawing>
What was the molecular weight of the protein(s) in the precipitate? What were the closest molecular weight protein(s) from the Gel Filtration lesson?
What was the molecular weight of the protein(s) in the supernatant? What were the closest molecular weight protein(s) from the Gel Filtration lesson?
The protein mixes analyzed in the Gel Filtration and this lesson on Protein Precipitaton are the same. Are all proteins accounted for in each lab? Explain.

Extra Credit
Give your own explanation for the precipitation of the protein(s). Include why some kinds of proteins precipitate and some do not.

Teacher Notes
Chemistry Concept: Hydrogen Bonding

To prepare saturated ammonium sulfate solution, add about 610 g (NH4)2SO4 to one liter of pure boiling water. Though saturation is achieved at only 4.1 M, you need this excess amount to compensate for volume expansion from the high mass concentration. Cool the SAS to room temperature to filter any crystals out. Store indefinitely.
The prescribed 4.0 ml SAS per 1.0 ml of serum mix brings the percentage saturation of ammonium sulfate to 80. You may want to experiment with the percent saturation depending on your protein mix. Albumins (including BSA) may not precipitate except at 70% or greater concentrations. Experiment to find the right concentration. BSA will precipitate well, above 75%.
If you redesign this lab to include a number of proteins in the mix, and if you choose to use BSA as one of the proteins, I have found I can dissolve up to 58 mg/ml in water. I use a concentration of 50 mg/ml.
Watch for centrifuge abuse! Reteach how to use a centrifuge, if necessary.
The answer to Processing the Data question number one is that the ammonium sulfate may interfere with gel filtration. It needs to be replaced with buffer upon dialysis. I recommend PBS 1X as the buffer. You may want to try 150 mM NaCl in distilled water so you can use the same buffer for the column as for the dialysis. To guard against cross-contamination of proteins, put each class's precipitate suspension in its own dialysis bag. Do the same with the supernatant. Allow the bags to spin in a bath of 150 mM NaCl buffer. Demonstrate again how you tie the bags. When you tie knots in each end, be careful to pull on the end and the knot, slipping the knot onto the bag rather than toward the end of the bag. Tightening in this way will prevent you from distorting the pore size of the membrane. Since students have experience tying the dialysis bag from the Gel Filtration lesson, you may allow a willing volunteer to tie the supernatant bag.
Some school districts (if not all) restrict the use of sodium azide. If this is true in your district, do not use sodium azide when preparing the beads.