Protein Technology Topics: Gel Filtration Chromatography

Gel filtration chromatography (also known as exclusion chromatography or molecular sieve method) is mainly based on the size and shape of the protein, that is, the quality of the protein is separated and purified. The packing in the column is some inert porous network structure material, mostly cross-linked polysaccharides, so that the substances in the protein mixture are separated according to the molecular size.

Experimental method protein gel filtration separation experimental material protein
Reagent, kit gel filtration buffer
Instrument, consumables, Buchner funnel gel filtration chromatography spectrophotometer

Experimental procedure

1. If the gel-filtered media is dry, it should be completely swollen in the gel filtration buffer.

2. Install the layered columns vertically on a stable laboratory stand in a constant temperature environment away from past passages and direct sunlight.

3. Using a syringe, inject the gel filtration buffer from the column's output tube into the column until the buffer reaches the column support plane and the syringe is left at the output to block the column.

4. Pour the gel suspension into the column to the height set along a glass rod placed along the inner wall of the column. Carefully add a 1 cm high buffer to the top of the gel and connect to the buffer storage container. Take the syringe that plugs the column output tube and wash the column with 2~3 times the bed volume of the buffer.

5. Run out of buffer on top of the column gel and close its output tube.

6. Add a protein sample equivalent to 1% to 5% of the bed volume. Open the output tube and let the sample flow into the column bed. The inner wall of the column above the gel is flushed with buffer.

7. Add a 1 cm high buffer layer to the top of the gel with a pipette and reconnect with the buffer storage container to begin elution.

8. The fraction collects the effluent. Each fraction is equivalent to 1% of the total bed volume, and each fraction of A280 is measured, and each sample is taken to measure biological activity, and the active fraction is selected to detect the quantity and quality of the protein contained by SDS-PAGE. , merge the fractions containing the target protein.

9. Wash the column with 2 to 3 bed volumes of gel filtration buffer. The column is stored in buffer containing 0.02% sodium azide and the column can be used indefinitely.
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Precautions Do not stir with a magnetic stirrer to allow the gel particles to settle naturally, aspirate or pour off fine particles that do not settle, or treat according to the manufacturer's instructions for use on pre-swelled gels in a Buchner funnel or glass. The preservative was removed from the fritted funnel with a large amount of buffer, resuspended in an equal volume of gel filtration buffer, poured into a narrow neck filter bottle, and degassed prior to use.

Computed Tomography

A CT scan makes use of computer-processed combinations of many X-ray images taken from different angles to produce cross-sectional (tomographic) images (virtual "slices") of specific areas of a scanned object, allowing the user to see inside the object without cutting.