Table 1. Resin Characteristics
|Bead Size||60-160 µm|
|Flow Rate1||>20 mL/min (>680 cm/hr) @ 25oC|
|Binding Capacity ||>5 mg of target protein/ ml of resin
|Storage Temperature||2-8o C|
|Chemical Stability2||Stable in all commonly used aqueous solutions and buffers.|
|Physical Stability2||Negligible volume variation due to changes in pH or ionic strength.|
1Linear flow rate = volumetric flow rate (cm3/h)/column cross-sectional area (cm2)
2Data refer to the coupled product, provided that the ligand can withstand the pH or chemical environment. Please note the following: pH stability, long term refers to the pH interval where the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. pH stability, short term refers to the pH interval for regeneration and cleaning procedures.
Instructions for Use
Wheat Germ Agglutinin (WGA) Superflow 4 is an affinity chromatography resin used for the purification of glycoproteins containing N-acetyl glucosamine (GlcNAc) groups or sialic acids. The immobilized ligand is a highly purified WGA lectin isolated from wheat germ (Triticum vulgaris). The resin is specifically used to purify insulin receptors, several serum and surface glycoproteins. WGA Superflow 4 is a highly stable resin and shows no leaching with commonly used buffers and in a pH range of 3-13. The resin can be used multiple times without the loss in yield and ligand activity.
WGA is a homodimeric lectin and has specificity for terminal GlcNAc and N-Acetylneuraminic acid (sialic acid) residues on glycoproteins. It preferentially binds to the dimeric or trimeric form of GlcNAc.
WGA Superflow 4 resin is supplied in as 50% slurry in PBS containing 20 Mm GlcNAc and 0.05% Sodium azide.
Upon receiving, store the resin at 2-8oC. Do not freeze.
Matrix support: 4% crosslinked agarose beads
Immobilized ligand: Highly purified Wheat Germ Agglutinin (WGA) lectin
Ligand density: 5 mg lectin/ml of agarose beads
Binding capacity: 3-5 mg of target protein/ml of resin
Storage buffer: PBS with 0.05% sodium azide.
Elution buffer: 0.5 M N-Acetyl-D-Glucosamine or Chitin Hydrolysate in PBS
Purification of GlcNAc/sialic acid containing glycoproteins from serum
WGA Superflow 4 resin can be packed in a disposable column (1-5 ml) and can also be used in a spin column depending on the scale of purification. Dilute the serum 1:1 in equilibration buffer (PBS).
1. Remove the resin from the fridge and equilibrate it at room temperature.
2. Pack the required amount of slurry in the column.
3. Equilibrate the packed column with 5 column volumes of PBS.
4. Apply the diluted serum on the column with a slow flow rate.
5. Wash the column with 5-10 bed volumes of PBS at high flow rate (until no protein is detected in the flowthrough, based on OD280).
6. Elute the bound glycoproteins with 0.5 M solution of N-Actyl-D-Glucosamine or chitin hydrolysate in PBS at slow flow rate and in multiple small fractions. If necessary, the pH of the elution buffer can be adjusted to pH 3.0 by acetic acid.
7. Measure the OD280 to determine the concentrated fractions. Run the SDS-PAGE and pool the fractions, dialyze in required buffer, concentrate, and store at required temperature.
8. The WGA Superflow 4 resin can be regenerated by washing the column with more than 20 column volumes of PBS. The clean column is then ready for another round of purification or can be stored at 4oC in buffer containing 0.05% of sodium azide.
To Download Instructions for use:
INST 937617SF4 WGA Superflow 4