SUN5 antibody

Catalog No. ABIN1876373

Product Details anti-SUN5 Antibody

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

Reactivity: Human, Mouse, Rat

Host: Rabbit

Clonality: Polyclonal

Conjugate: This SUN5 antibody is un-conjugated

Application: Western Blotting (WB)

Characteristics: Expected molecular weight of target: 379 kDa

Purification: Affinity purification

Isotype: IgG

Application Details

Application Notes: WB 1:500-1:2000

Restrictions: For Research Use only

Independent Validation (ICC)

Validation #102344 (Immunocytochemistry)

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

No.: #102344

Date: 12/19/2017

Antigen: SUN5

Method validated: Immunocytochemistry

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

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

Notes:

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.

Validation Images

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).

Full Methods

Primary Antibody: ABIN1876373

Secondary Antibody: 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.

Handling

Format: Liquid

Concentration: 1 mg/mL

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

Preservative: Sodium azide

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

Handling Advice: Avoid repeated freeze/thaw cycles.

Storage: -20 °C

Storage Comment: Store at -20°C.

Target Details for SUN5

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

Alternative Name: SPAG4L (SUN5 Products)

Synonyms: SPAG4L antibody, TSARG4 antibody, dJ726C3.1 antibody, 1700021O15Rik antibody, SPAG4L-2 antibody, SRG4 antibody, Spag4l antibody, Sad1 and UNC84 domain containing 5 antibody, SUN5 antibody, Sun5 antibody

Gene ID: 140732

UniProt: Q8TC36