DESCRIPTION:
Blue Sepharose Resin is a dye resin used in protein affinity chromatography (CRC Crit Rev Biochem. 1984; 16(2):169-205). For example, this resin was used to prepare the glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (G3PD), which was then used to treat and improve the texture of a meat product (Anim Sci J. 2011; 82(1):136-43). Blue Sepharose Resin has also been used to study other human phosphate binding proteins (Genet Mol Res. 2009; 8(3):929-37).
PROTOCOL:
Blue
Sepharose Resin is supplied preswollen in 20% ethanol, 0.1 M KH2PO4,
pH 8.0. Decant the 20% ethanol solution and replace it with binding buffer
(Typically low salt buffer such as 10 mM Sodium phosphate, pH 7.5). The binding
buffer should not contain agents that significantly increase the viscosity, but
the column may be equilibrated with viscous buffers at reduced flow rates after
packing is complete.
Column packing
1. Assemble the column (and packing reservoir if necessary).
2. Remove air from the end-piece and adapter by flushing with water. Make sure no air has been trapped under the column bed support. Close the column outlet leaving the bed support covered with water.
3. Resuspend the medium and pour the slurry into the column in a single continuous motion. Pouring the slurry down a glass rod held against the column wall will minimize the introduction of air bubbles.
4. If using a packing reservoir, immediately fill the remainder of the column and reservoir with water. Mount the adapter or lid of the packing reservoir and connect the column to a pump. Avoid trapping air bubbles under the adapter or in the inlet tubing.
5. Open the bottom outlet of the column and set the pump to run at the desired flow rate.
6. Maintain packing flow rate for at least 3 bed volumes after a constant bed height is reached. Mark the bed height on the column.
7. Stop the pump and close the column outlet.
8. If using a packing reservoir, disconnect the reservoir and fit the adapter to the column. With the adapter inlet disconnected, push the adapter down into the column until it reaches the mark. Allow the packing solution to flush the adapter inlet. Lock the adapter in position. Connect the column to a pump or a chromatography system and start equilibration. Re-adjust the adapter if necessary.
Binding
Proteins differ in their affinities for the Blue Sepharose resin. The resin capacity will also depend on parameters such as flow rate, pH, buffer composition and temperature.
1. Sample pH should be the same as that of the binding buffer. Filter the sample through a 0.22 µm or 0.45 µm filter to prolong the working life of the resin.
2. Load the sample.
3. Wash the medium with binding buffer until the baseline is stable.
Elution
1. Elution conditions vary with sample. Elution can be accomplished by a change in pH, polarity (e.g. by adding ethylene glycol) or ionic strength of the buffer. Enzymes can often be eluted at less than 1 M NaCl.
2. Competitive elution with low concentrations of the cofactor is required for very specifically bound proteins.
3. Either step or continuous gradients may be used.
STORAGE:
Long term storage (i.e. weeks): 4-8ºC in 20% ethanol, 0.1 M KH2PO4, pH 8.0. Do NOT freeze.
