SPAG4 anticorps

N° du produit ABIN4368846

Détail du produit anti-SPAG4 anticorps

Antigène: SPAG4 (Sperm Associated Antigen 4 (SPAG4))

Reactivité: Humain, Souris, Rat

Hôte: Lapin

Clonalité: Polyclonal

Conjugué: Cet anticorp SPAG4 est non-conjugé

Application: ELISA, Western Blotting (WB), Immunohistochemistry (IHC)

Information d'application

Restrictions: For Research Use only

Independent Validation (WB)

Validation #101449 (Western Blotting)

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

No.: #101449

Date: 31.08.2017

Antigène: SPAG4

Numéro du lot: 3522

Application validée: Western Blotting

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

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

Conclusion: ABIN4368846 specifically recognizes ectopically expressed SPAG4 and does not detect ectopic SPAG4L-2.

Validation Images

Different volumes of lysates from NIH/3T3 cells ectopically expressing SPAG4 (lanes 1-3) or SPAG4L-2 (lanes 5-7) were loaded and the blot membrane was incubated with ABIN4368846 (upper panel). Ectopically expressed proteins were subsequently detected using an anti-myc-tag antibody. Expected molecular masses of SPAG4 and SPAG4L-2 are approximately 50kDa.

NIH/3T3 cells expressing myc-tagged Spag4 (upper row) or Spag4L-2 (lower row) were incubated with an anti-myc tag antibody (left, red) or anti-SPAG4 antibody (middle, green). The pictures on the right show the red and green channels merged with DAPI counterstatin (blue).

Protocole

Anticorps primaire: ABIN4368846

Anticorps secondaire: anti-mouse IgG, HRP-linked (Sigma A9044, Lot 034M4761)

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 (Frohnert et al., 2011) expression plasmid using EndofectinMax (GeneCopoeia) following the manufacturer´s instructions.
• Grow cells for 24h.
• Lyse cells in SDS-sample buffer and denature total cellular lysates for 5min at 95°C.
• Separate cell lysates on a denaturing 10% SDS-PAGE gel (Laemmli 1970).
• Transfer proteins onto 0.2 µm Protran membrane (GE Healthcare, 10600004, A10043108) with a Western blotting system for 1h at 400mA (Towbin et al., 1979).
• Block the membrane in TBST (50mM Tris-HCl, pH7.4, 150mM NaCl, 0.2% Tween 20) containing 5% milk (blocking solution) for 60min at RT.
• Incubate membrane with primary rabbit anti-Sperm Associated Antigen 4 (SPAG4) antibody (antibodies-online, ABIN4368846, lot 3522) diluted 1:1000 in blocking solution ON at 4°C.
• Wash membrane with TBST for 30min at RT.
• Incubate membrane with secondary goat anti-rabbit IgG (H+L), HRP-linked (Jackson Immuno Research, 111-035-003, lot 123450) diluted 1:4000 in blocking solution for 45min at RT.
• Wash membrane 6x for 5 min with TBST.
• Reveal protein bands using Clarity Max Western ECL substrate (Bio-Rad, 1705062) and capture images via Chemidoc Imaging System (BioRad).
• Incubate membrane with primary mouse anti-myc tag antibody (clone 9E10) diluted 1:50 in blocking solution ON at 4°C.
• Wash membrane with TBST for 30min at RT.
• Incubate membrane with secondary anti-mouse IgG, HRP-linked (Sigma A9044, Lot 034M4761) diluted 1:5000 in blocking solution for 45min at RT.
• Reveal protein bands using Clarity Max Western ECL substrate (Bio-Rad, 1705062).

Notes: ABIN4368846 passes validation when used for detection of ectopically expressed proteins. However, it reacted with proteins specifically present in testicular tissue from day 7 post-partum to the adult and even in Spag4-deficient testis (Spag4-deficiency was verified by genotyping and RT-PCR on testis cDNA).

Independent Validation (ICC)

Validation #101455 (Immunocytochemistry)

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

No.: #101455

Date: 31.08.2017

Antigène: SPAG4

Numéro du lot: 3522

Application validée: Immunocytochemistry

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

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

Conclusion: The SPAG4 antibody ABIN4368846 specifically labels ectopically expressed Spag4 in cultured NIH/3T3 cells in ICC.

Validation Images

Different volumes of lysates from NIH/3T3 cells ectopically expressing SPAG4 (lanes 1-3) or SPAG4L-2 (lanes 5-7) were loaded and the blot membrane was incubated with ABIN4368846 (upper panel). Ectopically expressed proteins were subsequently detected using an anti-myc-tag antibody. Expected molecular masses of SPAG4 and SPAG4L-2 are approximately 50kDa.

NIH/3T3 cells expressing myc-tagged Spag4 (upper row) or Spag4L-2 (lower row) were incubated with an anti-myc tag antibody (left, red) or anti-SPAG4 antibody (middle, green). The pictures on the right show the red and green channels merged with DAPI counterstatin (blue).

Protocole

Anticorps primaire: ABIN4368846

Anticorps secondaire: 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 (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-Sperm Associated Antigen 4 (SPAG4) antibody (antibodies-online, ABIN4368846, lot 3522) diluted 1:100 in PBS ON at 4° and
  • mouse anti-myc tag antibody clone (9E10) diluted 1:50 in PBS ON at 4°C.
• Wash cells with TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 15min at RT.
• Incubate cells with secondary
• • goat anti-rabbit IgG DyLight488 (Thermo Scientific, 35553, lot KA130088) and
  • goat anti-mouse IgG (H+L) Alexa Fluor 555 (Invitrogen, A21422, lot 948498) diluted 1:2000 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 processsing using Adobe Photoshop 5.0.

Notes:

• An anti-myc antibody (red) detected both myc-tagged proteins, but anti the SPAG4 antibody ABIN4368846 (green) exclusively detected SPAG4 without any cross-reaction to SPAG4L-2. ABIN4368846 detected SPAG4 at the nuclear membrane, as expected.

• However, on mouse testicular cells ABIN4368846 decorated the manchette in spermatids (as was expected) but gave a much more intense signal on cells, that do not express Spag4 (Pasch et al., 2015)

Stockage

Format: Liquid

Concentration: 1.0 mg/mL

Agent conservateur: Sodium azide

Précaution d'utilisation: WARNING: Reagents contain sodium azide. Sodium azide is very toxic if ingested or inhaled. Avoid contact with skin, eyes, or clothing. Wear eye or face protection when handling. If skin or eye contact occurs, wash with copious amounts of water. If ingested or inhaled, contact a physician immediately. Sodium azide yields toxic hydrazoic acid under acidic conditions. Dilute azide-containing compounds in running water before discarding to avoid accumulation of potentially explosive deposits in lead or copper plumbing.

Stock: -20 °C

Détails sur SPAG4

Antigène: SPAG4 (Sperm Associated Antigen 4 (SPAG4))

Autre désignation: SPAG4 (SPAG4 Produits)

Synonymes: anticorps MGC133822, anticorps CT127, anticorps SUN4, anticorps 1700041K21Rik, anticorps MNCb-0953, anticorps Sun4, anticorps mKIAA4118, anticorps sperm associated antigen 4, anticorps SPAG4, anticorps Spag4

Sujet: Synonyms: Sperm associated antigen 4, SUN4, CT127