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SPAG4 antibody

SPAG4 Reactivity: Human, Mouse, Rat ELISA, WB, IHC Host: Rabbit Polyclonal unconjugated
Catalog No. ABIN4368846
  • Target See all SPAG4 Antibodies
    SPAG4 (Sperm Associated Antigen 4 (SPAG4))
    Reactivity
    Human, Mouse, Rat
    Host
    • 9
    • 2
    Rabbit
    Clonality
    • 9
    • 2
    Polyclonal
    Conjugate
    • 4
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    • 1
    This SPAG4 antibody is un-conjugated
    Application
    • 4
    • 4
    • 2
    • 1
    ELISA, Western Blotting (WB), Immunohistochemistry (IHC)
  • Restrictions
    For Research Use only
  • Validation #101449 (Western Blotting)
    'Independent Validation' Badge
    by
    Johann-Friedrich-Blumenbach-Institute for Zoology and Anthropology, Department of Developmental Biology, Georg-August-University Göttingen
    No.
    #101449
    Date
    08/31/2017
    Antigen
    SPAG4
    Lot Number
    3522
    Method validated
    Western Blotting
    Positive Control
    NIH/3T3 mouse embryonic fibroblast cells overexpressing HA- and myc-tagged SPAG4
    Negative Control
    NIH/3T3 mouse embryonic fibroblast cells overexpressing HA- and myc-tagged SPAG4L-2
    Notes
    ABIN4368846 specifically recognizes ectopically expressed SPAG4 and does not detect ectopic SPAG4L-2.
    'Independent Validation' Badge
    Validation Images
    Full Methods
    Primary Antibody
    ABIN4368846
    Secondary Antibody
    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).
    Experimental 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).
  • Validation #101455 (Immunocytochemistry)
    'Independent Validation' Badge
    by
    Johann-Friedrich-Blumenbach-Institute for Zoology and Anthropology, Department of Developmental Biology, Georg-August-University Göttingen
    No.
    #101455
    Date
    08/31/2017
    Antigen
    SPAG4
    Lot Number
    3522
    Method validated
    Immunocytochemistry
    Positive Control
    NIH/3T3 mouse embryonic fibroblast cells overexpressing HA- and myc-tagged SPAG4
    Negative Control
    NIH/3T3 mouse embryonic fibroblast cells overexpressing HA- and myc-tagged SPAG4L-2
    Notes
    The SPAG4 antibody ABIN4368846 specifically labels ectopically expressed Spag4 in cultured NIH/3T3 cells in ICC.
    'Independent Validation' Badge
    Validation Images
    Full Methods
    Primary Antibody
    ABIN4368846
    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 (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.
    Experimental 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)

  • Format
    Liquid
    Concentration
    1.0 mg/mL
    Preservative
    Sodium azide
    Precaution of Use
    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.
    Storage
    -20 °C
  • Target
    SPAG4 (Sperm Associated Antigen 4 (SPAG4))
    Alternative Name
    SPAG4 (SPAG4 Products)
    Synonyms
    MGC133822 antibody, CT127 antibody, SUN4 antibody, 1700041K21Rik antibody, MNCb-0953 antibody, Sun4 antibody, mKIAA4118 antibody, sperm associated antigen 4 antibody, SPAG4 antibody, Spag4 antibody
    Background
    Synonyms: Sperm associated antigen 4, SUN4, CT127
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