4006139A.
4006139A.qxd 6/23/98 8:11 AM Page B Table of Contents Section 1 Section 2 Section 3 3.1 3.2 3.3 Section 4 4.1 4.2 4.3 Section 5 Section 6 Page Introduction ..............................................1 Technical Description................................2 Principles of Gel Filtration ......................4 Gel Filtration Basics and Sample Preparation ..................................................4 Column Selection ........................................6 Eluant Selection ......................
4006139A.qxd 6/23/98 8:11 AM Page 1 Section 1 Introduction Bio-Gel A gels are a series of agarose based size exclusion gels which provide high resolving power, minimal non-specific interaction, and excellent flow properties. The porosity of the gel is controlled by the percentage of agarose incorporated into the matrix. Six fractionation ranges are available, with exclusion limits of 0.5, 1.5, 5, 15, and 50 million daltons. Bio-Gel A gels are compatible with all commonly used aqueous buffers.
006139A.qxd 6/23/98 8:11 AM Page 2 Table 2. Properties of Bio-Gel A Gels Section 2 Technical Description Product Grade Table 1. Bio-Gel A Gel Product Description Bio-Gel A-0.5m gel Matrix Bio-Gel agarose gel Particle size range Coarse Medium Fine 150–300 µm 75–150 µm 38–75 µm Shipping medium Hydrated Bead Typical Flow Fractionation Size Rates* Range/Exclusion (µm) (cm/hr) Limit (daltons) 50–100 mesh 100–200 mesh 200–400 mesh Fully hydrated in water, with 0.
4006139A.qxd 6/23/98 8:11 AM Page 4 Section 3 Principles of Gel Filtration Section 3.1 Gel Filtration Basics and Sample Preparation Gel filtration or size exclusion chromatography (SEC) separates molecules based on their size. The gel media consists of spherical beads containing pores of a specific size distribution. Separation occurs when molecules of different sizes are included or excluded from the pores within the matrix.
4006139A.qxd 6/23/98 8:11 AM Page 6 Section 3.2 Column Selection A properly executed separation will usually employ a bed with a length to diameter ratio between 5 and 10. The bed volume employed is usually 4 to 10 times the volume of the sample. The minimum anticipated dilution factor for an excluded substance approaches 1.25. Fractionation procedures generally require bed length to diameter ratios of 25 to 100 or greater and bed volumes 25 to 100 times the sample volume.
4006139A.qxd 4. 5. 6. 7. 8. 6/23/98 8:11 AM Page 8 be trapped in the bed support or the column end piece. Close the exit stopcock. Fit the column with a reservoir and filling funnel and add the gel slurry. Allow the gel to settle until it reaches a 3–5 cm bed height. Open the stopcock and allow buffer to flow until a stable gel bed has packed. Connect the flow adaptor tubing to the pump, fill it with buffer, and make sure it is free from air bubbles.
4006139A.qxd 6/23/98 8:11 AM Page 10 dextran is not recommended for Vo determination because it is not homogenous in size and may bind nonspecifically to the gel. Tobacco Mosaic Virus (TMV) or any large (>150 million) molecular weight marker may be used for Vo determination. Using a standard mixture of proteins allows verification of the column packing and protein elution as well as the calibration of the column and the calculation of the molecular weight of unknown sample proteins.
4006139A.qxd 6/23/98 8:11 AM Page 12 Section 6 Product Information Catalog Number Product Description 151-0130 151-0140 151-0150 Bio-Gel A-0.5m Gel, coarse, 500 ml Bio-Gel A-0.5m Gel, medium, 500 ml Bio-Gel A-0.5m Gel, fine, 500 ml 151-0430 151-0440 151-0450 Bio-Gel A-1.5m Gel, coarse, 500 ml Bio-Gel A-1.5m Gel, medium, 500 ml Bio-Gel A-1.
