HOXD8 Antikörper (C-Term)

Produktnummer ABIN1536919

Produktdetails anti-HOXD8 Antikörper

Target: HOXD8 (Homeobox D8 (HOXD8))

Bindungsspezifität: AA 243-270, C-Term

Reaktivität: Human

Wirt: Kaninchen

Klonalität: Polyklonal

Konjugat: Dieser HOXD8 Antikörper ist unkonjugiert

Applikation: Western Blotting (WB), Immunofluorescence (IF), Cleavage Under Targets and Release Using Nuclease (CUT&RUN)

Aufreinigung: This antibody is purified through a protein A column, followed by peptide affinity purification.

Immunogen: This HOXD8 antibody is generated from rabbits immunized with a KLH conjugated synthetic peptide between 243-270 amino acids from the C-terminal region of human HOXD8.

Klon: RB37165

Isotyp: Ig Fraction

Anwendungsinformationen

Applikationshinweise: IF: 1:10-50
WB: 1:1000
CUT&RUN: 1:100

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (CUT&RUN)

Validierung #104374 (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.: #104374

Datum: 26.04.2023

Antigen: HOXD8

Chargennummer: SA111222AR

Validierte Anwendung: Cleavage Under Targets and Release Using Nuclease

Positivkontrolle:

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

Negativkontrolle:

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

Bewertung:

Passed. The anti-HOXD8 antibody ABIN2850170 allows for CUT&RUN targeted profiling of HOXD8 in mouse forelimb cells.

Validierungsbilder

Library profiles comparing fragment size distributions on an E-Gel EX 2% agarose gel (Thermo Fisher). Fragments obtained from CUT&RUN using anti-HOXD8 antibody ABIN2850170 (right) after library preparation, compared to the E-Gel Sizing DNA Ladder (Thermo Fisher) (left).

1. Alignment tracks from CUT&RUN targeting HOXD8 in mouse fore limb (11.5) cells using anti-HOXD8 antibody ABIN2850170, showing the Cdc45 locus. 2. Alignment tracks using negative control IgG, ABIN101961. 3. RefSeq Genes.

Protokoll

Primärantikörper: ABIN2850170

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-HOXD8 ABIN2850170, 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.

Anmerkungen:

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

Handhabung

Format: Liquid

Buffer: Purified polyclonal antibody supplied in PBS with 0.09 % (W/V) sodium azide.

Konservierungsmittel: Sodium azide

Vorsichtsmaßnahmen: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Lagerung: 4 °C,-20 °C

Informationen zur Lagerung: HOXD8 Antibody (C-term) can be refrigerated at 2-8 °C for up to 6 months. For long term storage, keep at -20 °C.

Haltbarkeit: 6 months

Details zu HOXD8

Target: HOXD8 (Homeobox D8 (HOXD8))

Andere Bezeichnung: HOXD8 (HOXD8 Produkte)

Synonyme: HOX4 antikoerper, HOX4E antikoerper, HOX5.4 antikoerper, MGC68588 antikoerper, hox4 antikoerper, hox4e antikoerper, hox5.4 antikoerper, 4921540P06Rik antikoerper, AI047735 antikoerper, Hox-4.3 antikoerper, Hox-5.4 antikoerper, homeobox D8 antikoerper, homeobox D8 L homeolog antikoerper, HOXD8 antikoerper, hoxd8.L antikoerper, hoxd8 antikoerper, Hoxd8 antikoerper

Hintergrund: This gene belongs to the homeobox family of genes. The homeobox genes encode a highly conserved family of transcription factors that play an important role in morphogenesis in all multicellular organisms. Mammals possess four similar homeobox gene clusters, HOXA, HOXB, HOXC and HOXD, located on different chromosomes, consisting of 9 to 11 genes arranged in tandem. This gene is one of several homeobox HOXD genes located in a cluster on chromosome 2. Deletions that remove the entire HOXD gene cluster or the 5' end of this cluster have been associated with severe limb and genital abnormalities. In addition to effects during embryogenesis, this particular gene may also play a role in adult urogenital tract function.

Molekulargewicht: 31911

Gen-ID: 3234

NCBI Accession: NP_001186675, NP_062458

UniProt: P13378