SPAG4 antibody

Catalog No. ABIN4368846

Product Details anti-SPAG4 Antibody

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

Reactivity: Human, Mouse, Rat

Host: Rabbit

Clonality: Polyclonal

Conjugate: This SPAG4 antibody is un-conjugated

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

Application Details

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: 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.

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.

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).

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: 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.

Validation Images

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).

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)

Handling

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 Details for SPAG4

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