ODF2 antibody

Catalog No. ABIN5548695

Product Details anti-ODF2 Antibody

Target: ODF2 (Outer Dense Fiber of Sperm Tails 2 (ODF2))

Reactivity: Human, Rat, Mouse

Host: Rabbit

Clonality: Polyclonal

Conjugate: This ODF2 antibody is un-conjugated

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

Purification: Affinity purification

Immunogen: Recombinant protein of human ODF2

Isotype: IgG

Application Details

Application Notes: WB 1:500-1:2000, IHC 1:25-1:100

Restrictions: For Research Use only

Independent Validation (WB)

Validation #100023 (Western Blotting)

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

No.: #100023

Date: 05/03/2016

Antigen: Outer Dense Fiber of Sperm Tails 2 (ODF2)

Method validated: Western Blotting

Positive Control: recombinant ODF2-His, bacterially expressed; NIH3T3 total cell lysate

Notes: Passed, regarding sensitivity and specificity. The antibody detects bacterially expressed ODF2 (see 2)) and the endogenous protein (see 3)) with only moderate background staining in WB.

Validation Images

1. Positive control: protein ODF2-His, bacterially expressed, different amounts per lane, detection with anti His-Tag antibody (Novagen, 70796-3, Lot number N37164) (1:1,000). Secondary antibody anti mouse-HRP (Sigma-Aldrich A9044, Batch Number 034M4761) (1:10,000). 2. Test: protein ODF2-His, bacterially expressed, different amounts per lane, detection with anti ODF2 (ABIN2430582). 3.NIH3T3 total cell lysate (125µg/per lane), detection with anti ODF2 (ABIN2430582). Expected molecular weight of endogenous ODF2

Anti-ODF2 antibody ABIN2430582 (ESAP15572) is detected with a MFP488 goat anti-rabbit IgG (H+L) secondary antibody (green). The control mouse anti-γ-tubulin antibody is detected with a MFP590 goat anti-mouse secondary antibody (red). DNA is counterstained with DAPI (blue).

Full Methods

Primary Antibody: ABIN2430582

Secondary Antibody: anti rabbit IgG (H+L), HRP-linked (Jackson Immuno Research, Code number: 111-035-003, Lot number: 123450)

Full Protocol:

• Proteins separated on 10% SDS-PAGE (Laemmli 1970), blotted to Protran (GE Healthcare No. 10600004, 0.2 µm) (Towbin et al., 1979). Blocking of membrane in 5% skim milk in TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 30 min at room temperature.
• Incubation with first antibody in 5% skim milk in TBST at 4°C overnight.
• Washing in TBST for 30 minutes at room temperature.
• Incubation with secondary antibody in 5% skim milk in TBST for 45 minutes at room temperature. Washing in TBST for 30-45 minutes at room temperature.
• Secondary antibody: anti rabbit IgG (H+L), HRP-linked (Jackson Immuno Research, Code number: 111-035-003, Lot number: 123450). Dilution: 1:10,000.
• Chemiluminescence detection (Western Lightning Ultra, Lot 215-15091, Perkin Elmer) and image capture via BioRad Chemdoc or X-ray films.

Independent Validation (ICC)

Validation #100024 (Immunocytochemistry)

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

No.: #100024

Date: 05/03/2016

Antigen: Outer Dense Fiber of Sperm Tails 2 (ODF2)

Method validated: Immunocytochemistry

Positive Control: mouse anti-γ-tubulin (Sigma-Aldrich, clone GTU-88, T6557, lot number 072M4808)

Notes: Passed, the antibody detects centrosomal ODF2 in immunocytological preparations with only very low background staining (the green dots are mostly the correct location of ODF2 as they overlap with γ-tubulin spots (in red)).

Validation Images

1. Positive control: protein ODF2-His, bacterially expressed, different amounts per lane, detection with anti His-Tag antibody (Novagen, 70796-3, Lot number N37164) (1:1,000). Secondary antibody anti mouse-HRP (Sigma-Aldrich A9044, Batch Number 034M4761) (1:10,000). 2. Test: protein ODF2-His, bacterially expressed, different amounts per lane, detection with anti ODF2 (ABIN2430582). 3.NIH3T3 total cell lysate (125µg/per lane), detection with anti ODF2 (ABIN2430582). Expected molecular weight of endogenous ODF2

Anti-ODF2 antibody ABIN2430582 (ESAP15572) is detected with a MFP488 goat anti-rabbit IgG (H+L) secondary antibody (green). The control mouse anti-γ-tubulin antibody is detected with a MFP590 goat anti-mouse secondary antibody (red). DNA is counterstained with DAPI (blue).

Full Methods

Primary Antibody: ABIN2430582

Secondary Antibody: MFP488 goat anti-rabbit IgG (H+L) (MoBiTec, #MFP-A1008)

Full Protocol:

• NIH3T3 cells (ATCC) were grown on cover slips in DMEM, 10% fetal bovine serum, 5% penicillin/streptomycin (all Gibco) at 37°C in 5% CO2.
• Cells were fixed in ice-cold methanol for 20 min at -20°C.
• Unspecific binding sites were blocked in PBST (phosphate buffered saline (PBS) containing 0.15% bovine serum albumin, 0.1% Tween-20) for 1 hr.
• Cells were incubated with the primary antibodies at 4°C overnight.
• Cells were washed in TBST (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 15 min.
• Cells were incubated with secondary antibodies and DAPI for 1h at 37°C.
• Cells were washed in TBST for 15 min.
• Cells were embedded in Dako Fluorescent Mounting Medium (Dako).
• As positive control for centrosomal decoration anti ?-tubulin (Sigma-Aldrich, clone GTU-88, T6557, lot number 072M4808; 1:100) was used. anti ?-tubulin was detected with MFP590 goat anti-mouse IgG (H+L) (MoBiTec, #MFP-A1032; 1:300).
• Anti ODF2-antibody was detected with MFP488 goat anti-rabbit IgG (H+L) (MoBiTec, #MFP-A1008; 1:300).
• DNA was counterstained with DAPI.
• Images were taken by confocal microscopy (LSM 510, Zeiss), and processed using Adobe Photoshop 5.0.

Handling

Format: Liquid

Concentration: 0.3 mg/mL

Buffer: PBS with 0.05 % sodium azide and 50 % glycerol, PH7.4

Preservative: Sodium azide

Handling Advice: Avoid freeze / thaw cycles.

Storage: -20 °C

Storage Comment: Store at -20°C. Avoid freeze / thaw cycles.

References for anti-ODF2 antibody (ABIN5548695)

Rahimi, Cai, Kılıҫ, Kazerouni, Tapia Contreras, Hoyer-Fender: "Expression of α-Tubulin Acetyltransferase 1 and Tubulin Acetylation as Selective Forces in Cell Competition." in: Cells, Vol. 10, Issue 2, (2021) (PubMed).

Tapia Contreras, Hoyer-Fender: "CCDC42 Localizes to Manchette, HTCA and Tail and Interacts With ODF1 and ODF2 in the Formation of the Male Germ Cell Cytoskeleton." in: Frontiers in cell and developmental biology, Vol. 7, pp. 151 (PubMed).

Target Details for ODF2

Target: ODF2 (Outer Dense Fiber of Sperm Tails 2 (ODF2))

Alternative Name: ODF2 (ODF2 Products)

Synonyms: odf84 antibody, odf2/1 antibody, odf2/2 antibody, DKFZp469J0214 antibody, CT134 antibody, ODF2/1 antibody, ODF2/2 antibody, ODF84 antibody, si:dkey-228b2.4 antibody, AI848335 antibody, MMTEST29 antibody, outer dense fiber of sperm tails 2 antibody, outer dense fiber of sperm tails 2a antibody, outer dense fiber of sperm tails 2 L homeolog antibody, ODF2 antibody, odf2 antibody, odf2a antibody, odf2.L antibody, Odf2 antibody

Background: The outer dense fibers are cytoskeletal structures that surround the axoneme in the middle piece and principal piece of the sperm tail. The fibers function in maintaining the elastic structure and recoil of the sperm tail as well as in protecting the tail from shear forces during epididymal transport and ejaculation. Defects in the outer dense fibers lead to abnormal sperm morphology and infertility. This gene encodes one of the major outer dense fiber proteins. Alternative splicing results in multiple transcript variants.

Molecular Weight: Calculated MW: 95 kDa

Pathways: M Phase