PROCEDURE
Researchers are advised to optimize use of particles in any utility.
1. Put collectively 0.4mL of serum sample by diluting 1:20 with 10mM PBS.
2. Change 1mL of BioMag Plus Con A particles to a transparent microcentrifuge tube. Place the tube on a magnet to separate the particles from decision.
Rigorously take away and discard the reply.
3. Wash the particles by together with 1mL of Binding Buffer. Mix correctly.
4. Repeat the particle wash 1 further time. After the ultimate wash, take away the supernatant.
5. Add 0.1mL of Binding Buffer to the serum sample from Step 1. Add the sample to the particles and mix correctly by inversion or vortex to resuspend the particles.
6. Place the sample on a tube rotator and mix for 10-30 minutes at room temperature.
7. Take away the sample from the rotator and place in a magnetic separator. Rigorously take away the cleared supernatant.
8. Wash the particles by together with 0.5mL of Wash Buffer. Mix correctly 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 5 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. Mix 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 transparent microcentrifuge tube for later use or storage.
14. Repeat Steps 12-13. Eluates may be pooled and precipitated. Retailer eluates on ice for fast use or freeze for long-term storage.
DESCRIPTION
Bead- and column-based separation methods rely intently on the tempo and ease of affinity binding packages. Ligands resembling streptavidin, antibodies and lectins are used every to grab 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 useful for the labeling and isolation of glycanpresenting cells and glycoproteins in serum and cell lysate. Lectins have furthermore been utilized in cell adhesion analysis, to affect lymphocyte activation, and to find carbohydrate-based therapeutics.
Our Con A-coated BioMag Plus microparticles current a helpful means for isolating mannosyl- and glucosyl-containing glycoproteins and polysaccharides from serum or cell lysate, or for investigating completely different lectin / glycan-mediated processes. BioMag Concanavalin A has moreover been used for CUT&RUN, a chromatin profiling protocol that has quite a lot of key advantages over ChIP. (see References)
CHARACTERISTICS
Concanavalin A (Con A) is covalently related to functionalized BioMag Plus particles for use in glycoprotein isolations from serum and cell extracts. Con A is a 104,000 Da protein comprised of Four 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 groups 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. Each
binding web site requires calcium and manganese ions, and use of buffers with EDTA or completely different metallic chelators will result in an absence of carbohydrate binding ability.
Indicate diameter: ~1.0µm
Focus: 5 mg/mL
Con A certain: Determined by A280
concanavalin a
MATERIAL
Supplies Outfitted
3mL or 10mL of Con A coated particles in 10mM PBS with 0.1% sodium azide
Supplies Required
1.5mL or 2mL microcentrifuge tubes
Mammalian serum: 0.4mL of a 1:20 dilution in PBS / check out
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 methods 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
• Avoid the utilization of reagents with EDTA or completely different metallic chelators, as this could reduce the effectiveness of the Binding Buffer.
• Protease Inhibitors may be used when delicate glycoproteins are isolated.
• Low glycoprotein restoration may be related by each rising the elution incubation time previous 10 minutes, and / or by boiling particles in 200µL of
SDS-PAGE sample buffer for 5 minutes after which magnetically separating the particles from the eluate. (Remember: Boiling may detach some lectins and can
moreover launch nonspecifically certain proteins.)
• Run eluate samples on an SDS-PAGE 4-20% Tris-Glycine electrophoresis gel and stain the glycoprotein bands using the GlycoGel Stain Tools
(Polysciences’ Catalog Code 24693) to visualise.
• 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 visualise
completely different protein bands.
• The eradicating of albumin and IgG from serum samples may improve the isolation of low focus glycoproteins. If desired, use the BioMag® ProMax
Albumin Eradicating Tools (Catalog Code BP658) and / or the BioMag® ProMax Serum IgG Eradicating Tools (Catalog Code BP659).
