Gel Filtration

Background
Proteins are extracted from animals and humans as a mixture in a serum of body fluids. When immunologists want to study a specific protein, like an antibody, hormone, or enzyme, they need to separate it from the mix. One method of separating proteins, gel filtration, relies on the fact that proteins differ in molecular weights.

High molecular weight proteins will go down through a column swiftly, while lower molecular weight proteins take more time. This is because the structure of the gel beads within the column excludes molecules that are too big to pass through the bead pores. The sample mix of proteins is added to the top of the column along with a constant flow of buffer solution to keep the proteins moving. As the proteins flow past the beads, the smaller ones are free to enter through the pores. The excluded big molecules flow swiftly around the beads and thus, quickly exit the bottom of the column. The smaller proteins get there eventually, after passing through many pores on many beads.

An analogy is what happens when you throw a handful of rocks and dirt into a pool. The bigger, heavier rocks fall swiftly through the water, leaving the dirt to float slowly to the bottom. A better analogy is a system of lava rocks, water, and marbles. Imagine packing a wide tube with lava rocks. What would happen if you stood the tube upright and added to the top end a bucket of water mixed with marbles? Since lava rock is porous, you would expect the water to pass into it and trickle slowly down to the bottom of the tube. The marbles, on the other hand, would be too big to enter the pores. Instead, they would roll quickly along the outsides of the rocks, exiting the column first. In this experiment, the gel beads are the lava rocks, the small proteins are the water, and the large proteins are the marbles. (See figure.)

In this lab, you will separate a mixture of proteins found in bovine serum albumin, or BSA. You will begin by sending a mixture of dyes through a gel filtration column, collecting the elutes (run-off), and calibrating a curve to show how the column separates proteins by molecular weight. Then you will use the column to separate the BSA proteins. Next, you will add Branford assay reagent to indicate whether or not there is protein in each sample. Finally, you will use the calibration curve to identify the molecular weights of the proteins.

Procedure

Label fifteen small test tubes 1-15. Arrange them in order on a rack.
Prepare the gel bead column:
1. Clamp a 5¾ inch pipette to a lab stand. This will be your column.
2. Obtain a small glass wool plug. Do not touch it with your bare ands. Using a longer pipette, gently pack the plug into the bottom of the pipette body.
3. Use the long pipette to transfer beads into the column. Be gentle. You do not want to allow gaps or bubbles to form. It may help the beads to settle by flowing a small amount of buffer through the column between additions of beads. Make sure you catch the run-off with a small beaker. Fill leaving the top section empty. Use the groove as a fill line.
Obtain 10.0 µl of dye mix and add it to the top of the column. The dye will quickly enter the gel. As soon as it does, continue to add 150 mM NaCl buffer solution to the top.
Collect the elute, ten drops per tube, beginning with the tube labeled "1."
Record your observations. Indicate in which tubes the colors look the strongest.
Continue to add and collect the buffer in the run-off beaker as you prepare to run the BSA.
Rinse out the test tubes with tap water first, then distilled. Line the tubes back up for the next separation.
Obtain 10.0 µl of BSA and add it to the column.
Repeat steps three and four.
Test for protein presence. Add 30 µl of Branford assay reagent to each test tube. Cover the end and shake each tube back and forth (not up and down). If a bright blue color appears, there are proteins present in the sample. Record your observations, as in step five.
Clean up the column as follows: Gently invert and shake the beads into the recycle beaker for future use. Be patient. Some beads will come out the end. Then you can add some buffer solution and shake. The rest of the beads will eventually come out. Try slipping a pipette bulb over the thin end of the tube and onto the body. Try squeezing the bulb to back flush the beads out. Clean and return your pipette.
Dump your proteins and wash your test tubes.
Look through the microscope at the beads. Record your observations.

Processing the Data

Graph molecular weight versus volume fraction (tube number). This will be your calibration curve for the proteins. Use semi-log paper. The molecular weight of blue dextran is 2,000,000 Daltons. The molecular weight of phenol red is 378 Daltons. Connect the points with a straight line. If the colors are spread out over a range of volume fractions, find an average or peak intensity volume fraction.
How many proteins do you see?
What molecular weights should they have? Read Mw from the calibration
curve.
Describe how there may be more proteins than you think.
Describe how there may be fewer proteins than you think.
What is the very least number of proteins there could be?
If the blue dextran is composed solely of units of CH2, How many atoms would one molecule of blue dextran have? Show your calculations.
Likewise, how many atoms would your largest protein have?

Missing Figure. Molecular size separation by gel filtration.
(from Weir, D. M., Experimental Immunology, p. 37)

Teacher Notes

Chemistry Concept: Molecular weight

To prepare the calibrating dye mix, dissolve 0.025 g of blue dextran in 2 ml of a 50/50 mix of water and ethanol. Then, mix nine drops of the dextran to one drop of stock phenol red solution. The ten drops should provide over 200 µl of solution, more than enough for a class. Likewise, the two ml of blue dextran will be more than enough for five periods of chemistry.
I used Pharmacia's Sephadex G50 beads.
If you need a way of measuring 10 µl, have the students mark the stems of 5¾ inch pipettes twelve to thirteen milliliters up the stem. Filling to the line will give about 10 µl. A nine inch pipette should be marked nine to ten milliliters up the stem.
Use a 10 mg/ml concentration of BSA in PBS or 150 mM NaCl solution.
The column buffer is 150 mM NaCl in distilled water.
You should find tubes four through seven show the presence of protein This is one protein. The actual Mw of BSA protein is 70,000, which is in the range for volume fractions four through seven on the calibrated curve. If you take the average of those tubes (volume fraction 5.5) you will read 30,000 from the molecular weight, which is very close for the log scale. Taking a volume fraction of 5.0 gives a Mw of 60,000 Daltons.
Some school districts (if not all) restrict the use of sodium azide. If this is true in your district, do not use it when you prepare the beads.