Bead- and column-based separation methods relyheavily on the speed and ease of affinity binding systems. Ligands such asstreptavidin, antibodies and lectins are used both to capturespecifically-tagged targets and for the isolation of cells and biomoleculesthat naturally express the ligand binding partner. The uniquesaccharide-binding properties of plant lectins, such as Concanavalin A (Con A)have made them useful for the labeling and isolation of glycan presenting cellsand glycoproteins in serum and cell lysate. Lectins have additionally been usedin cell adhesion studies, to effect lymphocyte activation, and to explorecarbohydrate-based therapeutics. Concanavalin A magnetic beads has also beenused for CUT&RUN, a chromatin profiling protocol that has several keyadvantages over ChIP.
Store at 4°C for 2 years.
Researchers are advised tooptimize use of beads in any application
1.5mL or 2mL microcentrifugetubes
Binding Buffer:1x PBS + 1mM MgCl2 + 1mM MnCl2+
1mM CaCl2 (pH7.4)
Wash Buffer:1x PBS + 1mM MgCl2 + 1mM MnCl2+ 1mM CaCl2 (pH 7.4) + 0.1% Tween® 20
Elution Buffer;5mM Tris (pH 8.0) + 0.15M NaCl + 0.05% SDS + 1MGlucose
Preparation of Cells
1.Prepare mammalian cells（10000-100000 cells）sample，Centrifuge at 600 x g for 3-5 minuteat room temperature., Carefully remove the supernatant
2. Add 90ul of BindingBuffer to cells, Mix the tube to resuspend the cells. Centrifugeat 600 x g for 3-5 minute at room temperature, carefully remove the supernatant
3. Add 90μL of ElutionBuffer to the cells and resuspend carefully.
Preparation of Magnetic Beads
4. Transfer 10uL of Con A MagneticBeads to a clean microcentrifuge tube. Place the tube on a magnet to separatethe beads from solution.Carefully removeand discard the solution.
5. Wash the beads byadding 200μL of Binding Buffer. Mix well.
6. Repeat the particlewash 1 more time. After the last wash, remove the supernatant.
7. Add the cells sample from Step 3 to the beadsand mix well by inversion to resuspend the beads.
8. Place the sample on a tube rotator and mixfor 10-30 minutes at room temperature.
9. Remove the sample from the rotator and placein a magnetic separator. Carefully remove the cleared supernatant.
10. Wash the beads by adding 0.5mL of WashBuffer. Mix well by inversion or by vortex mixing.
11. Repeat Steps 9-10. Resuspend the beads with0.5mL of Wash Buffer and place on tube rotator for 5 minutes.
12. Repeat Steps 9-10.
13. Replace the tube of beads on the magneticseparator and carefully remove / discard the supernatant.
14. Add 50-250μL ofElution Buffer to the beads. Mix the tube to resuspendthe beads and place the tube on rotator for 10-30 minutes at room temperature.
15. Replace the tube of beads on the magneticseparator and carefully remove the eluate and transfer to a cleanmicrocentrifuge tube for later use or storage.
16. Repeat Steps 14-15. Eluates may be pooledand precipitated. Store eluates on ice for immediate use or freeze forlong-term storage.
• Avoid the use of reagentswith EDTA or other metal chelators, as this will reduce the effectiveness ofthe Binding Buffer.
•Protease Inhibitors may beused when sensitive glycoproteins are isolated.•Low glycoprotein recovery may be attached by either increasing the elutionincubation time beyond 10 minutes, and / or by boiling beads in 200μL of SDS-PAGEsample buffer for 5 minutes and then magnetically separating the beads from theeluate. (Note: Boiling may detach some lectins and may also release nonspecificallybound proteins.
ConA Magnetic Beads产品说明书