CHM655/755 Experiment 8
Purification of proteins by gel filtration chromatography Material:
-Sephadex™ G-75
-Tall/thin column
-Tubing clams
- 1 x TM buffer or other buffer systems dependent upon the target proteins to be parated.
-Fraction collection tubes
-Fraction collector
-Erlenmeyer flasks and beakers
-Parafilm
-Marker pens
-
Ruler
Proteins: lysozyme (14.7 kDa) and transferrin (75 kDa).
Procedure:
1.Determine the amount of resin to be ud. Bad on the information in the table
above, 1 g of G-75 dried resin can produce 12-15 ml of slurry in ddH2O.
2.Wash the resin extensively with ddH2O in a beaker. (Note: handle the resin gently
to prevent breakage of the fragile beads.)
3.Suspend the resin gently in ddH2O to a final volume that is twice the desired
packed bed volume.
4.Set up the column and make sure that the column is absolutely vertical. Clo the
outlet at the bottom of the column.
5.Place approximately one-tenth column volume of ddH2O into the empty column.
6.Pour the resin slurry into the column in one continuous motion. Do it slowly and
make sure the resin ttle down to the bottom without any air bubbles.
7.Let the resin stand for a few minutes, and then open the outlet at the bottom of the
column to allow flow of ddH2O through the column.
8.After the resin ttles, clo the outlet at the bottom of the column, and place a
column flow adapter on the top of the resin bed. It is important for gel filtration that the top of the slurry is even all around and no any bubbles are trapped inside the slurry packed in the column. Make sure that the upper flow adapter is filled with ddH2O before putting it onto the column; otherwi, bubbles will be introduced into the column from the flow adapter.
9.Before the sample is loaded to the column, it is necessary to equilibrate the
column with at least a 5 column volume of the buffer (1 x TM). Normally, the washing and elution buffers are the same. Carefully watch the washing buffer volume to avoid the column to be dried.
10.Set the fraction collector to 30-40 drops in each tube, producing approximately 2
ml of the eluate. Properly label the tubes for fraction collection.
11.Once completion of the column washing, clamp the bottom line and load the
protein mixture gently (10 mg/ml). After the sample (0.5-1.5 ml) completely gets into the resin, load about 2 ml of the elution buffer slowly on the top of the resin bed, then connect the top line to the elution buffer rervoir. Unclamp the bottom line and allow factions to be collected.
12.After completion of the collection, measure the O.D. value at 280 nm to obtain
chromatographs of the parated proteins. The eluates do not need to be diluted for measurement and calculate the concentration bad on the O.D. values. The samples then are placed at -20o C freezer for SDS-PAGE, western blot and other analys.
To test the properties of the column, it is a good practice to run a sample of 2mg/ml blue dextran on the newly poured column. Make a solution of 2mg/ml blue dextran in the chromatography buffer and filter to remove small, undissolved particles of blue dextran. Load the blue dextran in a volume of approximately 2% of the total column volume. Collect fractions using the automatic collector.
Protein Absorbance Assay (280 nm)
Considerations for u
Absorbance assays are fast and convenient, since no additional reagents or incubations are required. No protein standard need be prepared. The assay does not consume the protein. The relationship of absorbance to protein concentration is linear. Becau different proteins and nucleic acids have widely varying absorption characteristics there may be considerable error, especially for unknowns or protein mixtures. Any non-protein component of the solution that absorbs ultraviolet light will interfere with the assay. Cell and tissue fractionation samples often contain insoluble or colored components that interfere. The most common u for the absorbance assay is to monitor fractions from chromatography columns, or any time a quick estimation is needed and error in protein concentration is not a concern. An absorbance assay is recommended for calibrating bovine rum albumin or other pure protein solutions for u as standards in other methods.
Principle
Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm. Amino acids with aromatic rings are the primary reason for the absorbance peak at 280 nm. Peptide bonds are primarily responsible for the peak at 200 nm. Secondary, tertiary, and quaternary structure all affect absorbance, therefore factors such as pH, ionic strength, etc. can alter the absorbance spectrum.
Equipment
In addition to standard liquid handling supplies a spectrophotometer with UV lamp and quartz cuvette are required.
Procedure
Carry out steps 1-4 (280 nm only) for a very rough estimate. Carry out all steps if nucleic acid contamination is likely.
Warm up the UV lamp (about 15 min.)
Adjust wavelength to 280 nm
Calibrate to zero absorbance with buffer solution only
Measure absorbance of the protein solution
Adjust wavelength to 260 nm
Calibrate to zero absorbance with buffer solution only
Measure absorbance of the protein solution
Analysis
Unknown proteins or protein mixtures. U the following formula to roughly estimate protein concentration. Path length for most spectrometers is 1 cm.
Concentration (mg/ml) = Absorbance at 280 nm divided by path length (cm.)
Pure protein of known absorbance coefficient.U the following formula for a path length of 1 cm. Concentration is in mg/ml, %, or molarity depending on which type coefficient is ud.
concentration = Absorbance at 280 nm divided by absorbance coefficient
To convert units, u the relationships:
Mg protein/ml = % protein divided by 10 = molarity divided by protein molecular weight
Unknowns with possible nucleic acid contamination.U the following formula to estimate protein concentration:
Concentration (mg/ml) = (1.55 x A280) - 0.76 x A260)
Comments
Cold solutions can fog up the cuvette, while warm solutions can relea bubbles and interfere with the readings. For concentrated solutions (absorbance greater than 2) simply dilute the solution.
Absorbance coefficients of some common protein standards:
Bovine rum albumin (BSA): 63
Bovine, human, or rabbit IgG: 138
Chicken ovalbumin: 70
If the coefficient for a specific protein at a certain concentration is unknown, a standard curve is needed to accurately calculate the concentration of the protein.
