SALL4 Antikörper (AA 1-220)

Produktnummer ABIN6147364

Produktdetails anti-SALL4 Antikörper

Target: SALL4 (Sal-Like 4 (SALL4))

Bindungsspezifität: AA 1-220

Reaktivität: Human

Wirt: Kaninchen

Klonalität: Polyklonal

Konjugat: Dieser SALL4 Antikörper ist unkonjugiert

Applikation: Western Blotting (WB), Immunofluorescence (IF)

Sequenz: MSRRKQAKPQ HINSEEDQGE QQPQQQTPEF ADAAPAAPAA GELGAPVNHP GNDEVASEDE ATVKRLRREE THVCEKCCAE FFSISEFLEH KKNCTKNPPV LIMNDSEGPV PSEDFSGAVL SHQPTSPGSK DCHRENGGSS EDMKEKPDAE SVVYLKTETA LPPTPQDISY LAKGKVANTN VTLQALRGTK VAVNQRSADA LPAPVPGANS IPWVLEQILC

Kreuzreaktivität: Human, Maus

Produktmerkmale: Polyclonal Antibodies

Immunogen: Recombinant fusion protein containing a sequence corresponding to amino acids 1-220 of human SALL4 (NP_065169.1).

Isotyp: IgG

Anwendungsinformationen

Applikationshinweise: WB,1:500 - 1:2000,IF,1:50 - 1:100

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (CUT&RUN)

Validierung #104300 (Cleavage Under Targets and Release Using Nuclease)

by: Cantù Lab, Gene Regulation during Development and Disease, Linköping University

No.: #104300

Datum: 20.08.2021

Antigen: SALL4

Chargennummer: 29100201

Validierte Anwendung: Cleavage Under Targets and Release Using Nuclease

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

Negativkontrolle: Monoclonal anti-FLAG (Sigma-Aldrich, F3165)

Bewertung:

Passed. ABIN6132627 allows for SALL4 targeted digestion of genomic DNA using CUT&RUN.

Validierungsbilder

Alignment tracks from CUT&RUN targeting SALL4 in human melanoma cells. 1. Peaks called by SEACR from CUT&RUN data using subnucleosomal reads. 2. Alignment track for subnucleosomal CUT&RUN reads obtained using anti-SALL4 antibody ABIN6132627 in human melanoma cells. 3. Alignment track for all reads obtained through CUT&RUN using anti-SALL4 antibody ABIN6132627 in human melanoma cells. 4. Alignment track for negative control.

Protokoll

Primärantikörper: ABIN6132627

Full Protocol:

• Cell harvest
• • Harvest 500,000 human melanoma cells per antibody to be used at RT. /li>
  • Centrifuge cell solution 3 min at 600 x g at RT.
  • Remove the liquid carefully.
  • Gently resuspend cells in 1 mL Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free) by pipetting and transfer cell solution to a 2 mL microcentrifuge tube.
  • Centrifuge cell solution 3 min at 600 x g at RT and discard the supernatant.
  • Repeat twice for a total of three washes.
  • Resuspend cell pellet in 1 mL Wash Buffer by gently pipetting.
• Concanavalin A beads preparation
• • Prepare one 1.5 mL microcentrifuge tube.
  • Gently resuspend the magnetic Concanavalin A Beads (antibodies-online, ABIN6923139).
  • Pipette 10 µL Con A Beads slurry for each sample into the 1.5 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 each 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 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. 10 µL per sample.
• Cell immobilization – binding to Concanavalin A beads
• • Carefully vortex the cell suspension and add 10 µL of the Con A beads in Binding Buffer to the cell suspension for each sample.
  • Close tube tightly and rotate for 10 min at RT.
• Cell permeabilization and primary antibody binding
• • Divide cell suspension into separate 2 mL microcentrifuge tubes, one for each antibody (500,000 cells per sample).
  • Place the microcentrifuge tubes on a magnetic stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Place each tube at a low angle on the vortex mixer set to a low speed and add 150 µL Digitonin Wash buffer (wash buffer with 0.025% (wt/vol) Digitonin) supplemented with 2 mM EDTA.
  • Gently vortex the microcentrifuge tubes until the beads are resuspended.
  • Add 1.5 µL antibody (anti-SALL4 antibody ABIN6132627, anti-H3K27me3 positive control antibody ABIN6923144, and anti-FLAG tag antibody negative control) to the respective tube, corresponding to a 1:100 dilution.
  • Rotate the microcentrifuge tubes ON at 4 °C.
  • Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 ml pipette tip.
  • Repeat once for a total of two washes.
• pAG-MNase Binding
• • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Vortex the sample at low speed and add 3.75 µL 20X CUTANA pAG-MNase for ChIC/CUT&RUN Assays (ABIN6950951) to 0.5X in 150 µL Digitonin Wash Buffer per sample, gently resuspending the beads by pipetting.
  • Rotate the microcentrifuge tubes for 1 h at 4 °C.
  • Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tubes from the magnetic stand.
  • Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 mL pipette tip.
  • Repeat once for a total of two washes.
• MNase digestion and release of pAG-MNase-antibody-chromatin complexes
• • 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.
  • Place each tube at a low angle on the vortex mixer set to a low speed and add 100 μL Digitonin Wash buffer per sample along the side of the tube.
  • Place tubes in a heat block, kept on ice, and allow to chill.
  • Add 2 μL 0.1 M CaCl₂ to each sample.
  • Incubate tubes at 0 °C for 30 min.
  • Add 100 μL 2xSTOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% (wt/vol) Digitonin, 100 μg/mL RNAse A, 50 μg/mL Glycogen).
  • Incubate tubes at 37 °C for 30 min.
  • Place the tubes on a magnet stand until the fluid is clear.
  • Transfer the supernatant containing the pA-MNase-bound digested chromatin fragments to fresh 1.5 mL microcentrifuge tubes.
• DNA extraction
• • Add 2 µL 10% SDS to a final concentration of 0.1% and 2.5 µL Proteinase K (20 mg/mL) to each supernatant.
  • Gently vortex tubes at a low speed of approximately 1,100 rpm.
  • Incubate tubes at 50 °C for 1 h.
  • Add 200 µL PCI to tube.
  • Vortex tubes thoroughly at high speed until the liquid appears milky.
  • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at RT for 5 min.
  • Carefully transfer to upper aqueous phase to a fresh 1.5 mL microcentrifuge tube containing 2 µL glycogen (diluted 1:10 to 2 mg/mL from the 20 mg/mL stock solution).
  • Add 20 µL 3 M NaOAc pH 5.2.
  • Add 400 µL 100% ethanol.
  • Place tubes for at -20 °C ON.
  • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at 4 °C for 5min.
  • Remove the liquid carefully with a pipette.
  • Wash pellet with 1ml 70% ethanol.
  • Centrifuge tubes in a tabletop centrifuge at 16,000 x g at 4 °C for 1 min.
  • Remove the liquid carefully with a pipette.
  • Air-dry the pellet, then dissolve in 30 µL 1 mM Tris-HCl, 0.1 mM EDTA.
• 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), 36bp PE.
• Bioinformatics
• • Align reads the human genome (hg38) using bowtie78 with settings -X 700 -m1 -v 3. Remove duplicate reads, and sort files using samtools. Filter mapped reads for size, keeping only reads with a fragment size at or below 120 base pairs.
  • Generate bedgraph files using bedtools genomecov.
  • Call peaks using SEACR version 1.3, in relaxed mode, normalizing to the negative control.

Anmerkungen:

Results are published in Diener, J., Baggiolini, A., Pernebrink, M. et al. Epigenetic control of melanoma cell invasiveness by the stem cell factor SALL4. Nat Commun 12, 5056 (2021). https://doi.org/10.1038/s41467-021-25326-8

Handhabung

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.

Referenzen für anti-SALL4 Antikörper (ABIN6147364)

Diener, Baggiolini, Pernebrink, Dalcher, Lerra, Cheng, Varum, Häusel, Stierli, Treier, Studer, Basler, Levesque, Dummer, Santoro, Cantù, Sommer: "Epigenetic control of melanoma cell invasiveness by the stem cell factor SALL4." in: Nature communications, Vol. 12, Issue 1, pp. 5056, (2021) (PubMed).

Details zu SALL4

Target: SALL4 (Sal-Like 4 (SALL4))

Andere Bezeichnung: SALL4 (SALL4 Produkte)

Synonyme: SALL4 antikoerper, drrs antikoerper, sall4a antikoerper, znf797 antikoerper, MGC81541 antikoerper, hsal4 antikoerper, MGC76059 antikoerper, cb614 antikoerper, sb:cb372 antikoerper, sb:cb614 antikoerper, wu:fa01g03 antikoerper, ik:tdsubc_2c1 antikoerper, xx:tdsubc_2c1 antikoerper, 5730441M18Rik antikoerper, AA407717 antikoerper, AL022809 antikoerper, AW536104 antikoerper, C330011P20Rik antikoerper, C78083 antikoerper, C78563 antikoerper, Tex20 antikoerper, DRRS antikoerper, HSAL4 antikoerper, ZNF797 antikoerper, dJ1112F19.1 antikoerper, spalt like transcription factor 4 antikoerper, sal-like 4 (Drosophila) antikoerper, spalt-like transcription factor 4 L homeolog antikoerper, spalt-like transcription factor 4 antikoerper, SALL4 antikoerper, sall4 antikoerper, sall4.L antikoerper, Sall4 antikoerper

Hintergrund: This gene encodes a zinc finger transcription factor thought to play a role in the development of abducens motor neurons. Defects in this gene are a cause of Duane-radial ray syndrome (DRRS). Alternative splicing results in multiple transcript variants encoding different isoforms.,SALL4,DRRS,HSAL4,ZNF797,Epigenetics & Nuclear Signaling,Cell Biology & Developmental Biology,Stem Cells,Embryonic Stem Cells,SALL4

Molekulargewicht: 65 kDa/112 kDa

Gen-ID: 57167

UniProt: Q9UJQ4

Pathways: Stem Cell Maintenance, Tube Formation