ETV4 Antikörper (AA 1-207)
-
- Target Alle ETV4 Antikörper anzeigen
- ETV4 (Ets Variant 4 (ETV4))
-
Bindungsspezifität
- AA 1-207
-
Reaktivität
- Human
-
Wirt
- Kaninchen
-
Klonalität
- Polyklonal
-
Konjugat
- Dieser ETV4 Antikörper ist unkonjugiert
-
Applikation
- Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF)
- Sequenz
- MYLHTEGFSG PSPGDGAMGY GYEKPLRPFP DDVCVVPEKF EGDIKQEGVG AFREGPPYQR RGALQLWQFL VALLDDPTNA HFIAWTGRGM EFKLIEPEEV ARLWGIQKNR PAMNYDKLSR SLRYYYEKGI MQKVAGERYV YKFVCEPEAL FSLAFPDNQR PALKAEFDRP VSEEDTVPLS HLDESPAYLP ELAGPAQPFG PKGGYSY
- Kreuzreaktivität
- Human, Maus, Ratte
- Produktmerkmale
- Polyclonal Antibodies
- Aufreinigung
- Affinity purification
- Immunogen
- Recombinant fusion protein containing a sequence corresponding to amino acids 1-207 of human Pea3 / ETV4 (NP_001248368.1).
- Isotyp
- IgG
-
-
- Applikationshinweise
- WB,1:500 - 1:2000,IHC,1:50 - 1:200,IF,1:50 - 1:200
- Beschränkungen
- Nur für Forschungszwecke einsetzbar
-
- by
- Gianluca Zambanini, Anna Nordin and Claudio Cantù; Cantù Lab, Gene Regulation during Development and Disease, Linköping University
- No.
- #104387
- Datum
- 28.02.2022
- Antigen
- ETV4
- Chargennummer
- BA04368866
- 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. ABIN6131217 allows for ETV4 targeted digestion using CUT&RUN in mouse fore limb (11.5) cells.
- Primärantikörper
- ABIN6131217
- Sekundärantikörper
- 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.
- Add 2 µL antibody (anti-ETV4 antibody ABIN6131217, 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
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
Validierung #104387 (Cleavage Under Targets and Release Using Nuclease)ValidierungsbilderProtokoll -
- Format
- Liquid
- Buffer
- PBS with 0.02 % sodium azide,50 % glycerol, pH 7.3.
- Konservierungsmittel
- Sodium azide
- Vorsichtsmaßnahmen
- This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
- Lagerung
- -20 °C
- Informationen zur Lagerung
- Store at -20°C. Avoid freeze / thaw cycles.
-
- Target
- ETV4 (Ets Variant 4 (ETV4))
- Andere Bezeichnung
- ETV4 (ETV4 Produkte)
- Synonyme
- E1A-F antikoerper, E1AF antikoerper, PEA3 antikoerper, PEAS3 antikoerper, Pea3 antikoerper, AW414408 antikoerper, Pea-3 antikoerper, pea3 antikoerper, ETS variant 4 antikoerper, ets variant 4 antikoerper, ETV4 antikoerper, Etv4 antikoerper, etv4 antikoerper
- Hintergrund
- Transcriptional activator. May play a role in keratinocyte differentiation.,ETV4,E1A-F,E1AF,PEA3,PEAS3,ETS variant 4,Pea3/ETV4,Epigenetics & Nuclear Signaling,Transcription Factors,Neuroscience,ETV4
- Molekulargewicht
- 23 kDa/49 kDa/53 kDa
- Gen-ID
- 2118
- UniProt
- P43268
-