Magnetic Concanavalin A Beads (Agarose)

N° du produit ABIN6952467

Détail du produit

Highlights:

• Magnetic agarose concanavalin A beads for CUT&RUN and CUT&Tag assays.
• Superior handling compared to magnetic silica concanavalin A beads: easier washes and resuspension.
• Reduced risk for samples to dry out.
• Performance comparable to or better than magnetic silica concanavalin A beads.

Application: Cleavage Under Targets and Release Using Nuclease (CUT&RUN), Cleavage Under Targets and Tagmentation (CUT&Tag), Separation (Sep)

Fonction: Immobilization of whole cells through Concanavlin A binding of membrane glyocproteins.

Attributs du produit:

• Magnetic ConA Beads (Agarose) for CUT&RUN/CUT&Tag Assays are based on a ferrimagnetic core surrounded by an agarose matrix covalently bound via polyurethane links.
• In contrast to silica based beads containing superparamagnetic magnetite nanaoparticles, the hydrophilic surface of our Magnetic ConA Beads (Agarose) for CUT&RUN/CUT&Tag Assays reduces the risk of unspecific binding of contaminants. No residual charges are present after conjugation. This minimizes non-specific binding to the matrix.
• The weak magnetic moment does not interfere with their solubility in the absence of an external magnet field. Upon exposure to a magnetic field however, the beads show a stronger magnetic reaction than the superparamagnetic beads. They are therefore easier to pull out of a solution using a magnetic separator.
• The beads' diameter is with 30 μm considerably larger than for silica based ConA beads. Their capacity is comparable because of the open structure of the agarose carbohydrate network.

Ingrédients: 10% suspension of Concanavalin A coated agarose particles with a ferrimagnetic core

Bead Ligand: Concanavalin A

Bead Matrix: Magnetic Agarose beads

Bead Size: 30 μm

Information d'application

Indications d'application: Optimal working dilution should be determined by the investigator.

Commentaires:

• Metal ions (calcium and manganese) mediate the binding to Concanavalin A and stabilize is conformation.
• The use of buffers with EDTA or other metal chelators must be avoided as it will result in a loss of carbohydrate binding ability.

Protocole:

• Collect approximately 250,000 cells for each sample.
• Wash cells 3 times in 1 mL Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl).
• Take cells up in a volume of Wash Buffer corresponding to 250,000 cells/mL.
• Homogenize Magnetic ConA Beads (Agarose) for CUT&RUN/CUT&Tag Assays slurry by shaking.
• Take 10 μL bead slurry for each sample.
• Wash beads 3 times with 1 mL Binding Buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl₂, 1 mM MnCl₂.
• Resuspend beads in a volume of Binding Buffer corresponding to the initial volume of bead slurry.
• Add beads in Binding Buffer to the cells in Wash Buffer.
• Incubate for 30 min at RT to bind cells to Magnetic ConA Beads (Agarose) for CUT&RUN/CUT&Tag Assays.

Restrictions: For Research Use only

Independent Validation (CUT&RUN)

Validation #104288 (Cleavage Under Targets and Release Using Nuclease)

by: Max Planck Institut für Immunbiologie und Epigenetik

No.: #104288

Date: 24.08.2020

Antigène: ConA

Numéro du lot: cab0304001

Application validée: Cleavage Under Targets and Release Using Nuclease

Contrôle positif: Anti-H3K4me3 antibody

Contrôle négative: guinea pig anti-rabbit antibody ABIN101961

Conclusion:

Passed. ABIN6952467 allows immobilization of cells for CUT&RUN.

Validation Images

Fragment Analyzer profiles comparing fragment size distributions between reads obtained from CUT&RUN using an anti-H3K4me3 CUT&RUN Positive Control antibody in conjunction with silica-based magnetic ConA beads ABIN6923139 (left) or agarose-based magnetic ConA beads ABIN6952467 (right)

Alignment to mouse NCBI37/mm9 reference genome of reads from CUT&RUN targeting H3K4me3 in LSK cells using ABIN6952467 (tracks 3 and 4) and ENCODE H3K4me3 ChIP-seq signal (accession ENCFF001MZZ, track 2; peaks in track 1).

Protocole

Full Protocol:

• Cell harvest
• • Harvest 5,000 murine LSK cells per sample to be used at RT.
  • Centrifuge cell solution 3 min at 600 x g at RT.
  • Remove the liquid carefully.
  • Gently resuspend cells in 1 mL Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free) by pipetting and transfer cell solution to a 2 mL microcentrifuge tube.
  • Centrifuge cell solution 3 min at 600 x g at RT and discard the supernatant.
  • Repeat twice for a total of three washes.
  • Resuspend cell pellet in 1 mL Wash Buffer by gently pipetting.
• Concanavalin A beads preparation
• • Prepare one 1.5 mL microcentrifuge tube.
  • Gently resuspend the magnetic silica-based magnetic ConA beads ABIN6923139 or agarose-based magnetic ConA beads ABIN6952467.
  • Pipette 10 µL Con A Beads slurry for each sample into the 1.5 mL microcentrifuge tube.
  • Place the tube on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tube from the magnetic stand.
  • Pipette 1 mL Binding Buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl₂, 1 mM MnCl₂) into each tube and resuspend ConA beads by gentle pipetting.
  • Spin down the liquid from the lid with a quick pulse in a table-top centrifuge.
  • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tube from the magnetic stand.
  • Repeat twice for a total of three washes.
  • Gently resuspend the ConA Beads in a volume of Binding Buffer corresponding to the original volume of bead slurry, i.e. 10 µL per sample.
• Cell immobilization – binding to Concanavalin A beads
• • Carefully vortex the cell suspension and add 10 µL of the Con A beads in Binding Buffer to the cell suspension for each sample.
  • Close tube tightly and rotate for 10 min at RT.
• Cell permeabilization and antibody binding
• • Divide cell suspension into separate 2 mL microcentrifuge tubes, one for each antibody (5,000 cells per sample).
  • Place the microcentrifuge tubes on a magnetic stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Place each tube at a low angle on the vortex mixer set to a low speed and add 100 µL Digitonin Wash buffer (wash buffer with 0.025% (wt/vol) Digitonin) supplemented with 2 mM EDTA.
  • Gently vortex the microcentrifuge tubes until the beads are resuspended.
  • For the positive control, add 1 µL anti-H3K4me3 antibody to the respective tube, corresponding to a 1:100 dilution.
  • For the negative control, add 1 µL guinea pig anti rabbit negative control antibody (antibodies-online, ABIN101961, lot NE-200-12190001) to the respective tube, corresponding to a 1:100 dilution.
  • Rotate the microcentrifuge tubes ON at 4 °C.
  • Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 ml pipette tip.
  • Repeat once for a total of two washes.
• pA-MNase Binding
• • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Vortex the sample at low speed and add 150 μL pA-MNase solution at 700 ng/mL per sample, gently resuspending the beads by pipetting.
  • Rotate the microcentrifuge tubes for 1 h at 4 °C.
  • Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 mL pipette tip.
  • Repeat once for a total of two washes.
• MNase digestion and release of pA-MNase-antibody-chromatin complexes
• • Spin down the liquid from the lid with a quick pulse in a table-top centrifuge.
  • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Place each tube at a low angle on the vortex mixer set to a low speed and add 100 μL Digitonin Wash buffer per sample along the side of the tube.
  • Place tubes in a heat block, kept on ice, and allow to chill.
  • Add 2 μL 0.1 M CaCl₂ to each sample.
  • Incubate tubes at 0 °C for 30 min.
  • Add 100 μL 2xSTOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% (wt/vol) Digitonin, 100 μg/mL RNAse A, 50 μg/mL Glycogen).
  • Incubate tubes at 37 °C for 30 min.
  • Place the tubes on a magnet stand until the fluid is clear.
  • Transfer the supernatant containing the pA-MNase-bound digested chromatin fragments to fresh 1.5 mL microcentrifuge tubes.
• DNA extraction
• • Add 2 µL 10% SDS to a final concentration of 0.1% and 2.5 µL Proteinase K (20 mg/mL) to each supernatant.
  • Gently vortex tubes at a low speed of approximately 1,100 rpm.
  • Incubate tubes at 50 °C for 1 h.
  • Add 200 µL PCI to tube.
  • Vortex tubes thoroughly at high speed until the liquid appears milky.
  • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at RT for 5 min.
  • Carefully transfer the upper aqueous phase to a fresh 1.5 mL microcentrifuge tube containing 200 µL chloroform:isoamyl alcohol 24:1.
  • Vortex tubes thoroughly at high speed until the liquid appears milky.
  • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at 4 °C for 5 min.
  • Carefully transfer to upper aqueous phase to a fresh 1.5 mL microcentrifuge tube containing 2 µL glycogen (diluted 1:10 to 2 mg/mL from the 20 mg/mL stock solution).
  • Add 20 µL 3 M NaOAc pH 5.2.
  • Add 400 µL 100% ethanol.
  • Place tubes for at -20 °C ON.
  • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at 4 °C for 5min.
  • Remove the liquid carefully with a pipette.
  • Wash pellet with 1ml 70% ethanol.
  • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at 4 °C for 1 min.
  • Remove the liquid carefully with a pipette.
  • Air-dry the pellet, then dissolve in 30 µL 1 mM Tris-HCl, 0.1 mM EDTA.
• Library preparation and sequencing
• Read mapping and Peak calling

Notes:

• Transcription start sites were identified through CUT&RUN on LSK cells immobilized on magnetic ConA beads ABIN6952467 using an H3K4me3 antibody. Identified peaks were consistent with the Histone Mods by ChIP-seq from ENCODE (NCBI37/mm9).

• Background signal using the agarose based ConA beads ABIN6952467 appeared to be lower than in a parallel experiment using silica based ConA beads for cell immobilization.

Stockage

Format: Liquid

Buffer: 20 mM Sodium Acetate pH 6.6, 20% Ethanol

Conseil sur la manipulation: Do not freeze the Magnetic ConA Beads (Agarose) for CUT&RUN/CUT&Tag Assays!
Vortex bead suspension well before use.

Stock: 4 °C

Date de péremption: 12 months

Référence

Zambanini, Nordin, Jonasson, Pagella, Cantù: "A new cut&run low volume-urea (LoV-U) protocol optimized for transcriptional co-factors uncovers Wnt/b-catenin tissue-specific genomic targets." dans: Development (Cambridge, England), (2022) (PubMed).