SUN5 Antikörper

Produktnummer ABIN1876373

Produktdetails anti-SUN5 Antikörper

Target: SUN5 (Sad1 and UNC84 Domain Containing 5 (SUN5))

Reaktivität: Human, Maus, Ratte

Wirt: Kaninchen

Klonalität: Polyklonal

Konjugat: Dieser SUN5 Antikörper ist unkonjugiert

Applikation: Western Blotting (WB)

Produktmerkmale: Expected molecular weight of target: 379 kDa

Aufreinigung: Affinity purification

Isotyp: IgG

Anwendungsinformationen

Applikationshinweise: WB 1:500-1:2000

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (ICC)

Validierung #102344 (Immunocytochemistry)

by: Johann-Friedrich-Blumenbach-Institute for Zoology and Anthropology, Department of Developmental Biology, Georg-August-University Göttingen

No.: #102344

Datum: 19.12.2017

Antigen: SUN5

Validierte Anwendung: Immunocytochemistry

Positivkontrolle: NIH/3T3 mouse embryonic fibroblast cells overexpressing HA- and myc-tagged SPAG4L-2/SUN5

Negativkontrolle: NIH/3T3 mouse embryonic fibroblast cells overexpressing HA- and myc-tagged SPAG4

Bewertung:

Passed. ABIN1876373 is specific for SPAG4L-2/SUN5 and did not detect SPAG4 when ectopically expressed. Unspecific binding to other cellular proteins is nearly absent.

Validierungsbilder

Specificity of ABIN1876373. Cells were transfected either with SPAG4 (upper row) or SPAG4L-2/SUN5 (lower row), both tagged with HA and myc. Anti-myc antibody (B, F; red) detected both proteins, but ABIN1876373 (A, E; green) exclusively detected SPAG4L-2/SUN5 without any cross-reaction to SPAG4. The antibody detected SPAG4L-2/SUN5 at the nuclear membrane, as expected. The pictures on the right (D, H) show the red and green channels merged with DAPI counterstain (blue).

Protokoll

Primärantikörper: ABIN1876373

Sekundärantikörper: goat anti-rabbit IgG DyLight488 (Thermo Scientific, 35553, lot KA130088)

Full Protocol:

• NIH/3T3 cells (ATCC, CRL-1658) are grown in DMEM+GlutaMAX (Gibco, 31966-021, Lot 1852045) supplemented with fetal bovine serum (Gibco 270-106) and Pen/Strep (Gibco 15140), at 37°C and 5% CO₂ to 70% confluency.
• Transfect cells with HA/myc-tagged SPAG4 expression plasmid or HA/myc-tagged SPAG4L-2/SUN5 (Frohnert et al., 2011) expression plasmid using EndofectinMax (GeneCopoeia) following the manufacturer's instructions.
• Grow cells for 24h.
• Fix cells in 3.7% paraformaldehyde (in PBS) for 15min at 4°C followed by incubation in 0.3% Triton X-100 for 10min at 4°C.
• Block cells in PBS containing 1% bovine serum albumin and 0.5% Tween-20 (PBT) for 1h at room temperature.
• Incubate cells with primary
• • rabbit anti-SUN5 antibody (antibodies-online, ABIN1876373) diluted 1:100 in PBS ON at 4° and
  • mouse anti-myc tag antibody clone (9E10) diluted 1:100 in PBS ON at 4°C.
• Wash cells with TBST (50mM Tris-HCl, pH 7.4, 150mM NaCl, 0.1% Tween 20) for 15min at RT.
• Incubate cells with secondary
• • goat anti-rabbit IgG DyLight488 (Thermo Scientific, 35553, lot KA130088) diluted 1:1000 in PBS for 1h at 37°C and
  • goat anti-mouse IgG (H+L) Alexa Fluor 555 (Invitrogen, A21422, lot 948498) diluted 1:1000 in PBS for 1h at 37°C.
• Counterstain DNA with DAPI (4,6-Diamidino-2-phenylindole; Sigma D-9542).
• Image acquisition on Zeiss LSM 510 confocal microscope and processing using Adobe Photoshop 5.0.

Handhabung

Format: Liquid

Konzentration: 1 mg/mL

Buffer: PBS with 0.02 % sodium azide, 50 % glycerol, pH 7.4

Konservierungsmittel: Sodium azide

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

Handhabung: Avoid repeated freeze/thaw cycles.

Lagerung: -20 °C

Informationen zur Lagerung: Store at -20°C.

Details zu SUN5

Target: SUN5 (Sad1 and UNC84 Domain Containing 5 (SUN5))

Andere Bezeichnung: SPAG4L (SUN5 Produkte)

Gen-ID: 140732

UniProt: Q8TC36