HOXA13 antibody (AA 332-388)

Catalog No. ABIN1386749

Product Details anti-HOXA13 Antibody

Target: HOXA13 (Homeobox A13 (HOXA13))

Binding Specificity: AA 332-388

Reactivity: Human

Host: Rabbit

Clonality: Polyclonal

Conjugate: This HOXA13 antibody is un-conjugated

Application: Western Blotting (WB), ELISA, Immunofluorescence (Cultured Cells) (IF (cc)), Immunofluorescence (Paraffin-embedded Sections) (IF (p)), Immunohistochemistry (Frozen Sections) (IHC (fro)), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunocytochemistry (ICC)

Cross-Reactivity: Human

Predicted Reactivity: Mouse,Rat,Cow,Sheep,Pig,Horse,Chicken,Rabbit

Purification: Purified by Protein A.

Immunogen: KLH conjugated synthetic peptide derived from human HOXA13

Isotype: IgG

Application Details

Application Notes: WB 1:300-5000
ELISA 1:500-1000
IHC-P 1:200-400
IHC-F 1:100-500
IF(IHC-P) 1:50-200
IF(IHC-F) 1:50-200
IF(ICC) 1:50-200
ICC 1:100-500
CUT&RUN 1:100

Restrictions: For Research Use only

Independent Validation (CUT&RUN)

Validation #104368 (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.: #104368

Date: 04/26/2023

Antigen: HOXA13

Lot Number: AD02263837

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)

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-HOXA13 antibody ABIN1386749 (right) after library preparation, compared to the E-Gel Sizing DNA Ladder (Thermo Fisher) (left).

1. Alignment tracks from CUT&RUN targeting HOXA13 in mouse fore limb (11.5 DAC) cells using anti-HOXA13 antibody ABIN1386749, showing the Usp5 locus. 2. Alignment tracks using negative control IgG, ABIN101961. 3. RefSeq Genes.

Full Methods

Primary Antibody: ABIN1386749

Full Protocol:

• Cell harvest and nuclear extraction
• • Dissect 3 Fore limbs (11.5 DAC) from RjOrl:SWISS embryos 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 1.5 mL tube on the magnet rack 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, 2 mM EDTA).
  • Incubate for 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-HOXA13 antibody ABIN1386749, anti-H3K4me positive control antibody ABIN3023251, guinea pig anti-rabbit IgG negative control antibody ABIN101961) to the respective tube, corresponding to a 1:100 dilution.
  • Incubate ON 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 (to accelerate the process use a multichannel pipette).
  • Repeat the wash for a total of five washes.
• pAG-MNase Binding
• • Prepare a 1.5 mL microcentrifuge tube containing 200 µL of pAG mix pear sample (200 µL of wash buffer + 120 ng 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 200 µL of pAG-MNase premix.
  • Incubate for 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 for a total of five washes.
  • Resuspend in 200 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 51 µL of 2 mM CaCl₂ mix per sample (50 µL Wash Buffer + 1 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 50 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 move the supernatant in fresh collection tubes with 3 µL of EDTA/EGTA 0.25 M (Digestion buffer).
  • Resuspend the sample in 47 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 for 1 h at 4 °C.
  • Transfer the supernatant containing the pAG-MNase-bound digested chromatin fragments to the previously collected digestion buffer.
• DNA Clean up
• • Take the Mag-Bind® TotalPure NGS beads (Omega Bio-Tek, M1378-01) from the storage and wait until they are 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.
  • 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.
  • 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 and DNA in 20 µL of 10 mM Tris.
• 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 mm10 mouse 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. Development (2022). PMID 36355069

Handling

Format: Liquid

Concentration: 1 μg/μL

Buffer: 0.01M TBS( pH 7.4) with 1 % BSA, 0.02 % Proclin300 and 50 % Glycerol.

Preservative: ProClin

Precaution of Use: This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE, which should be handled by trained staff only.

Storage: 4 °C,-20 °C

Storage Comment: Shipped at 4°C. Store at -20°C for one year. Avoid repeated freeze/thaw cycles.

Expiry Date: 12 months

Target Details for HOXA13

Target: HOXA13 (Homeobox A13 (HOXA13))

Alternative Name: HOXA13 (HOXA13 Products)

Synonyms: HOXA13 antibody, RGD1562483 antibody, HOX1 antibody, HOX1J antibody, Hd antibody, Hox-1.10 antibody, homeobox A13 antibody, homeo box A13 antibody, HOXA13 antibody, Hoxa13 antibody

Background:

Synonyms: HOX1, HOX1J, Homeobox protein Hox-A13, Homeobox protein Hox-1J, HOXA13

Background: Sequence-specific, AT-rich binding transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis. Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis.

Gene ID: 3209

UniProt: P31271