Size Exclusion Chromatography (Gel Filtration Chromatography)
For more information see: Chapter 4 in Garett and Grisham or
Switzer and Garrity, Experimental Biochemistry,3rd ed WH Freeman and Co. 1999
SEC is a preparative, non-destructive analytical technique that permits the separation of molecules by their size. This is especially useful in protein purification because while there may be many proteins in a sample, their molecular weights can vary widely. This allows one to separate a mixture of proteins based on this wide size distribution. It also provides a simple procedure for removing salt (desalting) from a protein sample.
Molecules smaller than the exclusion limit of the gel material will become trapped in the gel beads. Those of larger molecular weight will not be trapped but will flow through the gel. The larger molecules are retarded only by their shape and their ease of passing by the beads. Thus, larger molecules elute first, while smaller molecules are held longer inside the beads and elute last.
Gel selection is very important. Gels are typically made of dextran (Sephadex), agarose (Sepharose) or polyacrylamide (Bio-Gel). Some gels, such as Spehadex G-10, retain only molecules of 0-700 daltons, while others, such as Sephadex G-200 retain molecules of 5000-600000 daltons.
In addition to the separation of a protein mixture based on size, gel filtration chromatography also allows one to estimate the molecular weight of an unknown globular protein. First, the column must be calibrated. To calibrate the column, one measures the “void volume” Vo of the column, i.e. the volume of liquid collected from the minute that the sample is applied to the column until an unretained molecule is eluted from the column (typically a large, dyed sugar such as blue dextran with a molecular weight of 2x106 daltons). This represents the volume that non-retained molecule experienced on its travels through the column. Each molecule will experience this volume, whether retained or not. This value must be measured each time a SEC column is prepared and used because it is unique to each column (based on packing density, gel composition, length etc.).
Second, a series of known molecular weight standard proteins are applied to the column and the volume at which each standard protein is eluted is recorded. This gives you Vr, the elution volume. A plot of molecular weight of the protein vs. Vr/Vo gives a calibration curve for the column. This is time consuming and the data for the calibration curve is unique to that column only. However, this is the most accurate way to obtain data from your SEC column.
In order to compare data between columns, a relative elution volume is used. Recall that Kd, the distribution constant describes how the protein is distributed between the two phases:
In SEC, this ratio can be expressed as a ratio of retention times or retention volumes (see your quantitative analysis book, chapter on chromatographic methods for the derivations).
Where Vr is the retention volume of the protein, Vo is the void volume and Vs is the volume of the stationary phase. The volume of the stationary phase is difficult to calculate accurately since the gel particles vary in size and volume in each batch and column. Instead, the average distribution constant is used:
where Vt is the maximum retention volume experienced by a small molecule such as a dye labeled amino acid. This is a close approximation of the stationary phase volume in the column. Kavg values have been determined for a number of gels and for a number of proteins. This allows one to compare Kavg values across different columns and different experiments. One can also plot log MW vs. Kd to get an estimate of the molecular weight of a protein. Remember, this is not as accurate as doing a full column calibration. One must be careful in interpreting these results. SEC separations are influenced by protein shape as well as ionic strength.
In this experiment, you will determine the void volume (Vo) and the total volume (Vt) of your SEC column. You will then run a calibration curve of your SEC column by separating a mixture of three standard proteins of known molecular weights. From their elution volume you will determine their Kavg. Finally, you will run an unknown protein sample and determine its Kavg and molecular weight.
How do we know when a protein is eluting from the column?
In the case of blue dextran and the dye labeled amino acid (any DNP amino acid), the molecules are colored blue or yellow respectively and one can see them elute from the column visually. However, most proteins do not exhibit a visual color (proteins containing hemes, like cytochrome –c or myoglobin will be red). Instead, they absorb at 280 nm with an extinction coefficient that is related to the number of aromatic amino acids in the protein (remember your three aromatic amino acids?). As you collect fractions, you will need to monitor EACH fraction on the UV-VIS at 280 nm. You should then plot Abs. vs. Volume to determine where the proteins elute, based on an increase in absorbance at 280 nm.
1.) You will prepare your column the week before the start of the lab as the gel needs time to settle.
2.) Obtain the SEC gel (G-100) from your instructor. It will already be soaking in 0.05 M pH 7 TRIS-HCl buffer with 0.1 M NaCl. This allows the beads to swell to their full size before pouring.
3.) Obtain a column from your instructor and plug the bottom with glass wool.
4.) Close the stop cock. Swirl the gel in the Erlenmeyer flask and pour it down a glass rod. It is VERY VERY VERY important that no air bubbles be trapped in your column (WHY?). If you have an air bubble, place parafilm on the top of the column, remove the column from the stand. Invert the column a number of times to redisperse the gel and then allow to resettle. If the air bubble is near the top of the column, you may be able to remove it with your glass rod.
5.) Pour the column at least 30-40 cm high.
6.) Open the stop cock and allow the buffer to pull through the column to just above the level of the gel.
7.) Close the stop cock. Cover the top with parrafilm and place in the refrigerator until the following week.
Determination of Void Volume (Vo) and Total Volume (Vt). Day 2
1.) Remove the column from the fridge and clamp it to the stand in a buret clamp.
2.) Obtain 250 mL of each of Blue dextran and DNP-amino acid in phosphate buffer.
3.) Mix the two samples to give a total volume of 0.5 mL.
4.) DO NOT LET THE COLUMN RUN DRY. Make sure that the level of the buffer at the top of the column is very close to the top level of the gel. If not, drain the buffer until it reaches the top of the gel.
5.) Using a long Pasteur pipette, add your 0.5 mL sample to the top of the gel with the stopcock closed. Do not disturb the gel at the top of the column.
6.) Place a graduated cylinder under the stopcock. Open the stopcock and allow the sample to move onto the column. Close the stop cock.
7.) Apply 1 mL of buffer to the top of the column.
8.) Drain into the graduated cylinder.
9.) Add 5-10 mL of buffer to the top of the column and drain into the graduated cylinder.
10.)Continue collecting volume into a graduated cylinder until the first appearance of blue color (from blue dextran) is seen to elute from the column. Record this total volume as accurately as possible (Vo).
11.)Collect all the blue dextran into the graduated cylinder. Remember to keep filling the buffer at the top of the column gently—do not disturb the level of the column)
12.)After all the blue dextran is past, record the total volume at this point.
13.)In a new graduated cylinder, collect volume from the time the blue dextran stops until the yellow DNP-amino acid elutes. Record this volume. Drain the column until all yellow dextran is removed. Vt is the total volume collected (blue dextran plus DNP-amino acid).
14.)Close the stopcock.
Standards (Obtaining Vr’s and Kd’s):
1.) Obtain a mixture of two protein standards. Record the name of the standards and their molecular weight. (myoglobin at 16,500 and bovine serum albumin at 67,000)
2.) Your elution order will be from largest to smallest. The proteins were chosen to try to cover the size exclusion range of the gel we are using.
3.) Apply 1 mL of the standard to the top of the column, again, taking care not to overly disturb the top of the column.
4.) Allow the sample to migrate onto the column by opening the stopcock. The eluting volume can be collected into a graduate cylinder up until 1 mL before the Vo determined in Part 1.
5.) Add 1 mL of buffer and collect the eluate.
6.) Add 5-10 mL at a time to the top of the column, taking care that it does not dry out!
7.) Collect your eluate in 1 mL marked test tubes beginning 1 mL before the Vo.
8.) Continue collecting fractions until you react Vt.
9.) Analyze your fractions on the UV-VIS. Be sure to run a blank of your buffer at 280 nm.
Run the Unknown protein:
1.) Absorbance vs. Volume and determine the volume where each of the three standard proteins eluted. Plot absorbance vs. volume for the unknown protein to determine where the unknown elutes.
2.) Plot MW vs. Vr/Vo for each protein (if you want you can also graph blue dextran and DNP amino acid.) Fit to a linear line in Excel and obtain the equation of the line.
3.) Calculate Kavg and plot log MW vs. Kavg for the protein samples. Fit to a linear line.
4.) Use the equations from each plot to determine the molecular weight of the unknown.