Concanavalin A (Con A) is covalently associated to functionalized BioMag Plus particles to be used in glycoprotein isolations from serum and cell extracts. Con A is a 104,000 Da protein comprised of 4 comparable subunits, and exists as an brisk dimer or tetramer relying upon pH. Its carbohydrate binding companions are α-D-glucose and α-D-mannose with unmodified OH teams at C-3, C-4, and C-6, and terminal glucose residues of proteins and peptides.
Con A agglutinates pink blood cells (RBCs), interacts with immunoglobulin glycopeptides, and is a lymphocyte mitogen. It binds some micro organism. Con A binding is mediated by metallic ions, which stabilize is conformation. Every
binding site requires calcium and manganese ions, and use of buffers with EDTA or utterly completely different metallic chelators will lead to an absence of carbohydrate binding means.
Point out diameter: ~1.0µm
Focus: 5 mg/mL
Con A sure: Decided by A280
PROCEDURE
Researchers are suggested to optimize use of particles in any utility.
1. Put collectively 0.4mL of serum pattern by diluting 1:20 with 10mM PBS.
2. Change 1mL of BioMag Plus Con A particles to a clear microcentrifuge tube. Place the tube on a magnet to separate the particles from choice.
Rigorously take away and discard the reply.
3. Wash the particles by along with 1mL of Binding Buffer. Combine accurately.
4. Repeat the particle wash 1 additional time. After the final word wash, take away the supernatant.
5. Add 0.1mL of Binding Buffer to the serum pattern from Step 1. Add the pattern to the particles and blend accurately by inversion or vortex to resuspend the particles.
6. Place the pattern on a tube rotator and blend for 10-30 minutes at room temperature.
7. Take away the pattern from the rotator and place in a magnetic separator. Rigorously take away the cleared supernatant.
8. Wash the particles by along with 0.5mL of Wash Buffer. Combine accurately by inversion or by vortex mixing.
9. Repeat Steps 7-8. Resuspend the particles with 0.5mL of Wash Buffer and place on tube rotator for five minutes.
10. Repeat Steps 7-9.
11. Substitute the tube of particles on the magnetic separator and punctiliously take away / discard the supernatant.
12. Add 250µL of Elution Buffer to the particles. Combine the tube to resuspend the particles and place the tube on rotator for 10-30 minutes at room temperature.
13. Substitute the tube of particles on the magnetic separator and punctiliously take away the eluate and swap to a clear microcentrifuge tube for later use or storage.
14. Repeat Steps 12-13. Eluates could also be pooled and precipitated. Retailer eluates on ice for quick use or freeze for long-term storage.
DESCRIPTION
Bead- and column-based separation strategies rely intently on the tempo and ease of affinity binding packages. Ligands resembling streptavidin, antibodies and lectins are used each to seize specifically-tagged targets and for the isolation of cells and biomolecules that naturally categorical the ligand binding companion.
The distinctive saccharide-binding properties of plant lectins, resembling Concanavalin A (Con A) have made them helpful for the labeling and isolation of glycanpresenting cells and glycoproteins in serum and cell lysate. Lectins have moreover been utilized in cell adhesion evaluation, to have an effect on lymphocyte activation, and to seek out carbohydrate-based therapeutics.
Our Con A-coated BioMag Plus microparticles present a useful means for isolating mannosyl- and glucosyl-containing glycoproteins and polysaccharides from serum or cell lysate, or for investigating utterly completely different lectin / glycan-mediated processes. BioMag Concanavalin A has furthermore been used for CUT&RUN, a chromatin profiling protocol that has numerous key benefits over ChIP. (see References)
concanavalin a
MATERIAL
Provides Outfitted
3mL or 10mL of Con A coated particles in 10mM PBS with 0.1% sodium azide
Provides Required
1.5mL or 2mL microcentrifuge tubes
Mammalian serum: 0.4mL of a 1:20 dilution in PBS / take a look at
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 strategies to ship 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.
concanavalin a cona
NOTES
• Keep away from the utilization of reagents with EDTA or utterly completely different metallic chelators, as this might scale back the effectiveness of the Binding Buffer.
• Protease Inhibitors could also be used when delicate glycoproteins are remoted.
• Low glycoprotein restoration could also be associated by every rising the elution incubation time earlier 10 minutes, and / or by boiling particles in 200µL of
SDS-PAGE pattern buffer for five minutes after which magnetically separating the particles from the eluate. (Keep in mind: Boiling might detach some lectins and may
furthermore launch nonspecifically sure proteins.)
• Run eluate samples on an SDS-PAGE 4-20% Tris-Glycine electrophoresis gel and stain the glycoprotein bands utilizing the GlycoGel Stain Instruments
(Polysciences’ Catalog Code 24693) to visualise.
• After GlycoGel staining, stain the gel utilizing Coomassie G250 (1mL or 2mL of 0.5% Coomassie G250 in 50% methanol and 10% acetic acid) to visualise
utterly completely different protein bands.
• The eradicating of albumin and IgG from serum samples might enhance the isolation of low focus glycoproteins. If desired, use the BioMag® ProMax
Albumin Eradicating Instruments (Catalog Code BP658) and / or the BioMag® ProMax Serum IgG Eradicating Instruments (Catalog Code BP659).
