TCF7 Antikörper (N-Term)

Produktnummer ABIN5620945

Dieses Anti-TCF7-Antikörper ist ein Kaninchen Polyklonal-Antikörper zur Detektion von TCF7 in WB, IHC, IP und CUT&RUN. Geeignet für Human. Unabhängig validiert für die Verwendung in Cleavage Under Targets and Release Using Nuclease. Dieses Primary Antibody wurde in 1 Publikation zitiert.

Kurzübersicht für TCF7 Antikörper (N-Term) (ABIN5620945)

Target: TCF7 (Transcription Factor 7 (T-Cell Specific, HMG-Box) (TCF7))

Reaktivität: Human

Wirt: Kaninchen

Klonalität: Polyklonal

Konjugat: Dieser TCF7 Antikörper ist unkonjugiert

Applikation: Western Blotting (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP), Cleavage Under Targets and Release Using Nuclease (CUT&RUN)

Produktdetails anti-TCF7 Antikörper

Bindungsspezifität: N-Term

Spezifität: Recognizes endogenous levels of TCF7 protein

Kreuzreaktivität (Details): Bovine

Produktmerkmale: Purified Polyclonal TCF7 antibody

Aufreinigung: TCF7 antibody was purified by immunogen affinity chromatography

Immunogen: TCF7 antibody was raised in Rabbit using a KLH-conjugated synthetic peptide encompassing a sequence within the N-term region of human TCF7 as the immunogen

Anwendungsinformationen

Applikationshinweise: The rabbit anti-TCF7 antibody ABIN5620945 is suitable for use in CUT&RUN, immunohistochemistry, immunoprecipitation, and Western Blot. Specific conditions for each assay should be optimized by the end user. General ABIN5620945 dilution recommendations for different applications are as follows:
IHC: 1:100-1:200
IP: 1:10-1:100
WB: 1:500-1:1,000
CUT&RUN: 1:100

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (CUT&RUN)

Validierung #104349 (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.: #104349

Datum: 28.02.2022

Antigen: TCF7

Chargennummer: X21031510

Validierte Anwendung: Cleavage Under Targets and Release Using Nuclease

Positivkontrolle:

Recombinant anti-H3K27me3 CUT&RUN Positive Control antibody (antibodies-online, ABIN6923144)

Negativkontrolle:

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

Bewertung:

Passed. ABIN5620945 allows for TCF7 targeted digestion using CUT&RUN in human HEK293T 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-TCF7 antibody ABIN5620945 after library preparation, compared to the E-Gel Sizing DNA Ladder (Thermo Fisher).

1. Alignment tracks from CUT&RUN targeting TCF7 in HEK293T cells using anti-TCF7 antibody ABIN5620945. 2 Peaks called by SEACR from CUT&RUN data using anti-TCF7 antibody ABIN5620945. 3. RefSeq Genes.

Protokoll

Primärantikörper: ABIN5620945

Full Protocol:

• Cell harvest and nuclear extraction
• • Harvest 250,000 HEK293T cells per antibody to be used at RT stimulated with 10 µM CHIR for 24 h at RT.
  • Centrifuge cell solution 5 min at 600 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.
  • Add 2 µL antibody (anti-TCF7 antibody ABIN5620945, anti-H3K27me3 antibody positive control ABIN6923144, 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.
  • 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.
• 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.

Anmerkungen:

Results are 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

Handhabung

Format: Liquid

Buffer: Supplied in liquid form in 0.42 % Potassium phosphate, 0.87 % Sodium chloride, pH 7.3 with 30 % glycerol and 0.01 % 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: Store at 4 deg C for short term storage. For long term, aliquot and store at -20 deg C. Avoid repeat freeze/thaw cycles

Referenzen für anti-TCF7 Antikörper (ABIN5620945)

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." in: Development (Cambridge, England), (2022) (PubMed).

Details zu TCF7

Target: TCF7 (Transcription Factor 7 (T-Cell Specific, HMG-Box) (TCF7))

Andere Bezeichnung: TCF7

Pathways: WNT Signalweg