The annexin family of calcium-binding proteins contains several family members that are characterized by a conserved core domain which binds phospholipids in a Ca2+-dependent manner, and a unique amino-terminal region which may confer binding specificity. Annexin family members have been implicated as regulators of such diverse processes as ion flux, endocytosis, exocytosis and cellular adhesion. Annexin A10, also known as ANX14 or ANXA10, is a 324 amino acid protein that contains four Annexin domains and may be involved in the regulation of cellular growth and signal transduction pathways throughout the cell. The gene encoding Annexin A10 maps to human chromosome 4, which encodes nearly 6% of the human genome and has the largest gene deserts (regions of the genome with no protein encoding genes) of all of the human chromosomes.
Background References
1. Morgan R.O., et al. 1999. Novel human and mouse annexin A10 are linked to the genome duplications during early chordate evolution. Genomics 60:40-49.
2. The MGC Project Team. 2004. The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). Genome Res. 14:2121-2127.
Western blot analysis of Annexin A10 on different lysates with Rabbit anti-Annexin A10 antibody (ET1610-57) at 1/1,000 dilution.
Lane 1: HeLa cell lysate (20 µg/Lane) Lane 2: PC-3 cell lysate (20 µg/Lane) Lane 3: Mouse colon tissue lysate (40 µg/Lane) Lane 4: Rat colon tissue lysate (40 µg/Lane)
Predicted band size: 37 kDa Observed band size: 35 kDa
Exposure time: 8 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1610-57) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Immunohistochemical analysis of paraffin-embedded human stomach tissue with Rabbit anti-Annexin A10 antibody (ET1610-57) at 1/1,000 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1610-57) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded mouse stomach tissue using anti-Annexin A10 antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1610-57, 1/200) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded rat bladder tissue using anti-Annexin A10 antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1610-57, 1/200) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded rat stomach tissue using anti-Annexin A10 antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1610-57, 1/200) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunocytochemistry analysis of HeLa cells labeling Annexin A10 with Rabbit anti-Annexin A10 antibody (ET1610-57) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Annexin A10 antibody (ET1610-57) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
Flow cytometric analysis of HeLa cells labeling Annexin A10.
Cells were fixed and permeabilized. Then stained with the primary antibody (ET1610-57, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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