How to load gel filtration column experiment?
How to load gel filtration column experiment?
Summary
This experiment describes how to load a gel filtration column. This experiment is from Protein Purification and Identification Laboratory Guide by Houzhu Zhu.
Operation method
How to load gel filtration column experiment
Materials and Instruments
XK26 40 columns SephacrylS-300 HR Blue Dextran TM Buffer + 0.1 mol LKCl
Move
Materials and equipment
XK26/40 column (PharmaciaBiotech, Inundefined)
SephacrylS-300 HR (PharmaciaBiotech, Inc.)
Blue Dextran
Reagents
TM buffer + 0.1 mol/L KCl
(For the recipe, see "Preparation of reagents", PP.131-138.)
Operational anomalies
1) Determine the amount of filler. The commercially available Sephacryl S-300 HR Gel is a slurry with a consistency of 2/3 of the packed bed volume. Therefore, for a 180 ml column (diameter = 2.6 cm, length = 34 cm), 270 ml of S-300 slurry can be removed from the bottle.
Note: When loading a gel filtration column, the homogeneity of the packed bed is very important. It should also be noted that thinner and longer columns are much more effective for high resolution separations.
2) Sufficiently wash the S-300 gel with several times the volume of TM buffer + 0.1 mol/L KCl in a coarse sintered glass funnel.
Note: Handle gently (especially when stirring) to prevent breakage of the fragile gel particles.
3) Gently suspend the filler in TM Buffer + 0.1 mol/L KCl until the final volume is twice the desired volume of the filled bed (360 ml).
4) (This step is optional but recommended) Degas the slurry of S-300 gel under vacuum for several minutes. This process removes dissolved gases from the media and buffer.
5) Stand up the column with the reservoir for the S-300 gel and buffer. Make sure the column is absolutely vertical. Close the outlet at the bottom of the column.
6) Add approximately 1/10 of the column volume of Buffer (approximately 20 ml) to the empty column.
7) Fill the column continuously with a slurry of S-300 gel.
Note: It is important to prevent air bubbles from entering the gel filtration column, as even small air bubbles can destroy the column, and if so, the column must be reloaded.
8) Allow the column to stand for about 30 min, then open the outlet at the bottom of the column and allow the buffer to flow through the column.
9) After the S-300 gel has settled, close the outlet at the bottom of the column, remove the reservoir from the column, and connect the column flow-through connector at the top of the packed bed. Note that it is important to fill the top flow-through fitting with buffer before attaching it to the column, otherwise air bubbles will be introduced from this flow-through fitting.
10) Connect the upper flow-through connector to the pump (beware of air bubbles) and begin to pressurize the column with the pump. Load the column at the flow rate used for chromatography (100 ml/h for the column in this experiment). When loading and pressing, watch the top of the packing and as the top of the packing sinks, adjust the upper liquid flow joint downward so that it is always at the top of the packed bed. Then, when the packing stops sinking, increase the flow rate to 150% of the chromatographic flow rate (i.e., for an S-300 column, increase the flow rate to 150 ml/h). Continue to press the column (and adjust the upper flow fitting until the packing is no longer further compressed. At this point the column is loaded and ready for use.
Note: When loading the column, it is not necessary to add protease inhibitors and dithiothreitol to the buffer. However, the column must be equilibrated with at least one full volume of buffer containing these components before the sample is loaded onto the column.
11) A good way to check column performance is to chromatograph a 2 mg/ml sample of blue dextran on a freshly loaded column. Do not chromatograph blue dextran on a column that was originally loaded with a protein sample. Prepare a 2 mg/ml solution of blue dextran in Chromatography Buffer (TM Buffer + 0.1 mol/L KCl) and filter (remove small, insoluble blue dextran particles with a 0.22um membrane). Blue dextran was sampled at approximately 2% of the total column volume (approximately 4 ml for the S-300 column) and the fractions were collected in 2.5 ml tubes. Chromatography with blue dextran provides information on (i) the empty volume, (ii) the degree of dilution of the sample, and (iii) the overall mass of the column, which can be determined by visual observation of the downward movement of the blue dextran in the column, thus revealing deficiencies in the column bed.
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