DESCRIPTION

Bead- and column-based separation methods rely heavily on the speed and ease of affinity binding systems. Ligands such as streptavidin, antibodies and  lectins are used both to capture specifically-tagged targets and for the isolation of cells and biomolecules that naturally express the ligand binding partner.  The unique saccharide-binding properties of plant lectins, such as Concanavalin A (Con A) have made them useful for the labeling and isolation of glycan presenting cells and glycoproteins in serum and cell lysate. Lectins have additionally been used in cell adhesion studies, to effect lymphocyte activation, and  to explore carbohydrate-based therapeutics

Our Con A-coated BioMag?Plus microparticles provide a convenient means for isolating mannosyl- and glucosyl-containing glycoproteins and  polysaccharides from serum or cell lysate, or for investigating other lectin / glycan-mediated processes. BioMag? Concanavalin A has also been used for  CUT&RUN, a chromatin profiling protocol that has several key advantages over ChIP. (see References)

CHARACTERISTICS

Concanavalin A (Con A) is covalently attached to functionalized BioMag?Plus  particles for use in glycoprotein isolations from serum and cellular extracts.  Con A is a 104,000 Da protein comprised of four identical subunits, and exists  as an active dimer or tetramer depending upon pH. Its carbohydrate binding  partners are α-D-glucose and α-D-mannose with unmodified OH groups at  C-3, C-4, and C-6, and terminal glucose residues of proteins and peptides.  Con A agglutinates red blood cells (RBCs), interacts with immunoglobulin  glycopeptides, and is a lymphocyte mitogen. It binds some bacteria.  Con A binding is mediated by metal ions, which stabilize is conformation. Each  binding site requires calcium and manganese ions, and use of buffers with EDTA  or other metal chelators will result in a loss of carbohydrate binding ability.

Mean diameter: ~1.0μm

Concentration: 5 mg/mL

Con A bound: Determined by A280

MATERIAL

Material Supplied

Determined by A280  3mL or 10mL of Con A coated particles in 10mM PBS with 0.1% sodium azide

Material Required

1.5mL or 2mL microcentrifuge tubes

Mammalian serum: 0.4mL of a 1:20 dilution in PBS / test  Binding Buffer: 1x PBS + 0.1% NaN3  + 1mM MgCl2  + 1mM MnCl2  + 1mM CaCl2  (pH 7.4)

Wash Buffer: 1x PBS + 0.1% NaN3  + 1mM MgCl2  + 1mM MnCl2  + 1mM CaCl2  (pH 7.4) + 0.1% Tween? 20

Con A particle Elution Buffer: 5mM Tris (pH 8.0) + 0.15M NaCl + 0.05% SDS + 1M Glucose

Precision pipets with disposable tips to deliver 20-200μL, 200-1000μL

Microcentrifuge Tube Separator:1.5mL Magnetic Separator (Catalog Code LS001)BioMag? Multi-6 Microcentrifuge Tube Separator (Catalog Code MS002)  Vortex mixer and tube rotator

NOTES

? Avoid the use of reagents with EDTA or other metal chelators, as this will reduce the effectiveness of the Binding Buffer

? Protease Inhibitors may be used when sensitive glycoproteins are isolated.

? Low glycoprotein recovery may be attached by either increasing the elution incubation time beyond 10 minutes, and / or by boiling particles in 200μL of  SDS-PAGE sample buffer for 5 minutes and then magnetically separating the particles from the eluate. (Note: Boiling may detach some lectins and may  also release nonspecifically bound proteins.)

? Run eluate samples on an SDS-PAGE 4-20% Tris-Glycine electrophoresis gel and stain the glycoprotein bands using the GlycoGel Stain Kit  (Polysciences’ Catalog Code 24693) to visualize.

? After GlycoGel staining, stain the gel using Coomassie G250 (1mL or 2mL of 0.5% Coomassie G250 in 50% methanol and 10% acetic acid) to visualize  other protein bands.

? The removal of albumin and IgG from serum samples may improve the isolation of low concentration glycoproteins. If desired, use the BioMag? ProMax  Albumin Removal Kit (Catalog Code BP658) and / or the BioMag? ProMax Serum IgG Removal Kit (Catalog Code BP659).

STORAGE AND STABILITY 

Store at 2-8?C. Freezing, drying, or centrifuging particles may result in irreversible aggregation and loss of binding activity.

SAFETY

This particle suspension contains sodium azide. Sodium azide may react with lead and copper plumbing to form explosive metal azides. Upon disposal of  material, flush with a large volume of water to prevent azide accumulation. Please consult the Safety Data Sheet for more information.

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