GLI3 antibody

Catalog No. ABIN2855813

Product Details anti-GLI3 Antibody

Target: GLI3 (GLI Family Zinc Finger 3 (GLI3))

Reactivity: Human

Host: Rabbit

Clonality: Polyclonal

Conjugate: This GLI3 antibody is un-conjugated

Application: Western Blotting (WB), Immunofluorescence (IF), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunocytochemistry (ICC)

Cross-Reactivity: Human, Mouse, Rat

Characteristics: Rabbit Polyclonal antibody to GLI3 (GLI family zinc finger 3)
GLI3 antibody

Purification: Purified by antigen-affinity chromatography.

Immunogen: Recombinant protein encompassing a sequence within the center region of human GLI3. The exact sequence is proprietary.

Isotype: IgG

Application Details

Application Notes: WB: 1:500-1:3000. ICC/IF: 1:100-1:1000. IHC-P: 1:100-1:1000. Optimal dilutions/concentrations should be determined by the researcher. Not tested in other applications.

Comment:

Positive Control: Jurkat , mouse ESC , PC-12 , Rat2

Restrictions: For Research Use only

Independent Validation (CUT&RUN)

Validation #104394 (Cleavage Under Targets and Release Using Nuclease)

by: Gianluca Zambanini, Anna Nordin and Claudio Cantù; Cantù Lab, Gene Regulation during Development and Disease, Linköping University

No.: #104394

Date: 09/08/2022

Antigen: GLI3

Lot Number: 40716

Method validated: Cleavage Under Targets and Release Using Nuclease

Positive Control:

Polyclonal rabbit anti-H3K4me (antibodies-online, ABIN3023251)

Negative Control:

Polyclonal guinea pig anti-rabbit IgG (antibodies-online, ABIN101961)

Notes:

Passed. ABIN2855813 allows for GLI3 targeted digestion using CUT&RUN in mouse fore limb (11.5) cells.

Validation Images

Library profiles comparing fragment size distributions on an E-Gel EX 2% agarose gel (Thermo Fisher). Fragments obtained from CUT&RUN using anti-GLI3 antibody ABIN2855813 and anti-H3K4me antibody ABIN3023251 after library preparation, compared to the E-Gel Sizing DNA Ladder (Thermo Fisher) (to the left of each).

1. Alignment tracks from CUT&RUN targeting H3K4me in mouse fore limb (11.5) cells using anti-H3K4me antibody ABIN3023251. 2. Alignment tracks from CUT&RUN targeting GLI3 in mouse fore limb (11.5) cells using anti-GLI3 antibody ABIN2855813, showing GLI target gene Ptch1. 3. RefSeq Genes.

Full Methods

Primary Antibody: ABIN2855813

Full Protocol:

• Cell harvest and nuclear extraction
• • Dissect 3 Fore limbs (11.5 DAC) from mouse strain RjOrl:SWISS for each sample.
  • Dissociate the tissue into single cells in TrypLE for 15 min at 37 °C.
  • Centrifuge cell solution 5 min at 800 x g at RT.
  • Remove the liquid carefully.
  • Gently resuspend cells in 1 mL of Nuclear Extraction Buffer (20 mM HEPES-KOH pH 8.2, 20% Glycerol, 0.05% IGEPAL, 0.5 mM Spermidine, 10 mM KCl, Roche Complete Protease Inhibitor EDTA-free).
  • Move the solution to a 2 mL centrifuge tube.
  • Pellet the nuclei 800 x g for 5 min.
  • Repeat the NE wash twice for a total of three washes.
  • Resuspend the nuclei in 20 µL NE Buffer per sample.
• Concanavalin A beads preparation
• • Prepare one 2 mL microcentrifuge tube.
  • Gently resuspend the magnetic Concanavalin A Beads (antibodies-online, ABIN6952467).
  • Pipette 20 µL Con A Beads slurry for each sample into the 2 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 the 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 the wash 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. 20 µL per sample.
• Nuclei immobilization – binding to Concanavalin A beads
• • Carefully vortex the nuclei suspension and add 20 µL of the Con A beads in Binding Buffer to the cell suspension for each sample.
  • Close tube tightly incubates 10 min at 4 °C.
  • Put the 2 mL tube on the magnet stand and when the liquid is clear remove the supernatant.
  • Resuspend the beads in 1 mL of EDTA Wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free, 2mM EDTA).
  • Incubate 5 min at RT.
  • Place the tube on the magnet stand and when the liquid is clear remove the supernatant.
  • Resuspend the beads in 200 µl of Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free) per sample.
• Primary antibody binding
• • Divide nuclei suspension into separate 200 µL PCR tubes, one for each antibody (150,000 cells per sample).
  • Add 2 µL antibody (anti-GLI3 antibody ABIN2855813, anti-H3K4me positive control antibody ABIN3023251, and guinea pig anti-rabbit IgG negative control antibody ABIN101961) to the respective tube, corresponding to a 1:100 dilution.
  • Incubate at 4 °C ON.
  • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Wash with 200 µL of Wash Buffer using a multichannel pipette to accelerate the process.
  • Repeat the wash five times for a total of six washes.
• pAG-MNase Binding
• • Prepare a 1.5 mL microcentrifuge tube containing 100 µL of pAG mix per sample (100 µL of wash buffer + 58.5 µg pAG-MNase per sample).
  • Place the PCR tubes with the sample on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove tubes from the magnetic stand.
  • Resuspend the beads in 100 µL of pAG-MNase premix.
  • Incubate 30 min at 4 °C.
  • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Wash with 200 µL of Wash Buffer using a multichannel pipette to accelerate the process.
  • Repeat the wash five times for a total of six washes.
  • Resuspend in 100 µL of Wash Buffer.
• MNase digestion and release of pAG-MNase-antibody-chromatin complexes
• • Place PCR tubes on ice and allow to chill.
  • Prepare a 1.5 mL microcentrifuge tube with 102 µl of 2 mM CaCl₂ mix per sample (100 µl Wash Buffer + 2 µL 100 mM CaCl₂) and let it chill on ice.
  • Always in ice, place the samples on the magnetic rack and when the liquid is clear remove the supernatant.
  • Resuspend the samples in 100 µl of the 2 mM CaCl₂ mix and incubate in ice for exactly 30 min.
  • Place the sample on the magnet stand and when the liquid is clear remove the supernatant.
  • Resuspend the sample in 50 µl of 1x Urea STOP Buffer (8.5 M Urea, 100 mM NaCl, 2 mM EGTA, 2 mM EDTA, 0.5% IGEPAL).
  • Incubate the samples 1h at 4°C.
  • Transfer the supernatant containing the pAG-MNase-bound digested chromatin fragments to fresh 200 µl PCR tubes.
• DNA Clean up
• • Take the Mag-Bind® TotalPure NGS beads (Omega Bio-Tek, M1378-01) from the storage and wait until they are at RT.
  • Add 2x volume of beads to each sample (e.g. 100 µL of beads for 50 µL of sample).
  • Incubate the beads and the sample for 15 min at RT.
  • During incubation prepare fresh EtOH 80%.
  • Place the PCR tubes on a magnet stand and when the liquid is clear remove the supernatant.
  • Add 200 µl of fresh 80% EtOH to the sample without disturbing the beads (Important!!! Do NOT resuspend the beads or remove the tubes from the magnet stand or the sample will be lost).
  • Incubate 30 sec at RT.
  • Remove the EtOH from the sample.
  • Repeat the wash with 80% EtOH.
  • Resuspend the beads in 25 µL of 10 mM Tris-HCl pH 8.2.
  • Incubate the sample for 2 min at RT.
  • Repeat the 2x beads clean up as described before (this time with 50 µL of beads for each sample).
  • Resuspend the beads + DNA in 20 µL of 10 mM Tris-HCl pH 8.2.
• Library preparation and sequencing
• • Prepare Libraries using KAPA HyperPrep Kit using KAPA Dual-Indexed adapters according to protocol.
  • Sequence samples on an Illumina NextSeq 500 sequencer, using a NextSeq 500/550 High Output Kit v2.5 (75 Cycles), 36 bp PE.
• Peak calling
• • Trim reads using using bbTools bbduk (BBMap - Bushnell B. - sourceforge.net/projects/bbmap/) to remove adapters, artifacts and repeat sequences.
  • Map aligned reads to the hg38 human genome using bowtie with options -m 1 -v 0 -I 0 -X 500.
  • Use SAMtools to convert SAM files to BAM files and remove duplicates.
  • Use BEDtools genomecov to produce Bedgraph files.
  • Call peaks using SEACR with a 0.001 threshold and the option norm stringent.

Experimental Notes:

The protocol is published in Zambanini, G. et al. A New CUT&RUN Low Volume-Urea (LoV-U) protocol uncovers Wnt/β-catenin tissue-specific genomic targets. bioRxiv (2022). https://doi.org/10.1101/2022.07.06.498999

Handling

Format: Liquid

Concentration: 1 mg/mL

Buffer: 1XPBS ( pH 7), 1 % BSA, 20 % Glycerol, 0.01 % Thimerosal

Preservative: Thimerosal (Merthiolate)

Precaution of Use: This product contains Thimerosal (Merthiolate): a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: 4 °C,-20 °C

Storage Comment: Store as concentrated solution. Centrifuge briefly prior to opening vial. For short-term storage (1-2 weeks), store at 4°C. For long-term storage, aliquot and store at -20°C or below. Avoid multiple freeze-thaw cycles.

Target Details for GLI3

Target: GLI3 (GLI Family Zinc Finger 3 (GLI3))

Alternative Name: GLI family zinc finger 3 (GLI3 Products)

Synonyms: ACLS antibody, GCPS antibody, GLI3-190 antibody, GLI3FL antibody, PAP-A antibody, PAPA antibody, PAPA1 antibody, PAPB antibody, PHS antibody, PPDIV antibody, xgli3 antibody, AI854843 antibody, AU023367 antibody, Bph antibody, Pdn antibody, Xt antibody, add antibody, im:7142603 antibody, GLI3 antibody, GLI family zinc finger 3 antibody, GLI family zinc finger 3 L homeolog antibody, GLI-Kruppel family member GLI3 antibody, GLI3 antibody, Gli3 antibody, gli3.L antibody, gli3 antibody

Background: This gene encodes a protein which belongs to the C2H2-type zinc finger proteins subclass of the Gli family. They are characterized as DNA-binding transcription factors and are mediators of Sonic hedgehog (Shh) signaling. The protein encoded by this gene localizes in the cytoplasm and activates patched Drosophila homolog (PTCH) gene expression. It is also thought to play a role during embryogenesis. Mutations in this gene have been associated with several diseases, including Greig cephalopolysyndactyly syndrome, Pallister-Hall syndrome, preaxial polydactyly type IV, and postaxial polydactyly types A1 and B.

Cellular Localization: Nucleus

Molecular Weight: 170 kDa

Gene ID: 2737

UniProt: P10071

Pathways: Hedgehog Signaling