PRODUCT CODE: em1801-11

APE1 Monoclonal Antibody (EM1801-11)

Applications

  • WB

  • ICC

  • IHC-P

  • FC

REACTIVITY

  • Human

  • Mouse

Western blot analysis of APE1 on HL-60 lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody was used at a 1:1,000 dilution in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:5,000 dilution was used for 1 hour at room temperature.
  • Western blot analysis of APE1 on HL-60 lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody was used at a 1:1,000 dilution in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:5,000 dilution was used for 1 hour at room temperature.
  • ICC staining APE1 in A549 cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with APE1 monoclonal antibody at a dilution of 1:50 for 1 hour at room temperature, washed with PBS. Alexa Fluorc™ 488 Goat anti-Mouse IgG was used as the secondary antibody at 1/100 dilution. The nuclear counter stain is DAPI (blue).
  • ICC staining APE1 in SiHa cells (green). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with APE1 monoclonal antibody at a dilution of 1:50 for 1 hour at room temperature, washed with PBS. Alexa Fluorc™ 488 Goat anti-Mouse IgG was used as the secondary antibody at 1/100 dilution. The nuclear counter stain is DAPI (blue).
  • Immunohistochemical analysis of paraffin-embedded human liver tissue using anti-APE1 antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 20 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the antibody (EM1801-11) at 1/200 dilution, for 30 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chrogen. Counter stained with hematoxylin and mounted with DPX.
  • Immunohistochemical analysis of paraffin-embedded human prostate cancer tissue using anti-APE1 antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 20 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the antibody (EM1801-11) at 1/200 dilution, for 30 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chrogen. Counter stained with hematoxylin and mounted with DPX.
  • Immunohistochemical analysis of paraffin-embedded human kidney tissue using anti-APE1 antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 20 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the antibody (EM1801-11) at 1/200 dilution, for 30 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chrogen. Counter stained with hematoxylin and mounted with DPX.
  • Immunohistochemical analysis of paraffin-embedded mouse colon tissue using anti-APE1 antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 20 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the antibody (EM1801-11) at 1/200 dilution, for 30 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chrogen. Counter stained with hematoxylin and mounted with DPX.
  • Flow cytometric analysis of APE1 was done on SiHa cells. The cells were fixed, permeabilized and stained with APE1 antibody at 1/100 dilution (red) compared with an unlabelled control (cells without incubation with primary antibody; black). After incubation of the primary antibody on room temperature for an hour, the cells was stained with a Alexa Fluor™ 488-conjugated goat anti-mouse IgG Secondary antibody at 1/500 dilution for 30 minutes.
Western blot analysis of APE1 on HL-60 lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody was used at a 1:1,000 dilution in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:5,000 dilution was used for 1 hour at room temperature.

Applications

  • WB

  • ICC

  • IHC-P

  • FC

REACTIVITY

  • Human

  • Mouse

SPECIFICATIONS

Product Type

Mouse monoclonal primary

Product Name

APE1 Monoclonal Antibody (EM1801-11)

Immunogen

Recombinant protein within human ape1 aa 20-350.

Host

Mouse

Positive Control

HL-60, A549, human liver tissue, human prostate cancer tissue, human kidney tissue, mouse colon tissue, SiHa.

Conjugation

Unconjugated

Clonality

Monoclonal

Clone Number

12H1

PROPERTIES

Form

Liquid

Storage Condition

Store at +4C after thawing. Aliquot store at -20C. Avoid repeated freeze / thaw cycles.

Storage Buffer

1*PBS (pH7.4), 0.2% BSA, 50% Glycerol. Preservative: 0.05% Sodium Azide.

Concentration

2 ug/ul

PURIFICATION

Protein affinity purified.

MOLECULAR WEIGHT

36 kDa

Isotype

IgG2a

APPLICATION DILUTION

  • WB:1:1,000-1:5,000

  • ICC:1:50 IHC-P:1:50-1:200

  • FC:1:50-1:100

TARGET

UNIPROT #

PROTEIN NAME

DNA-(apurinic or apyrimidinic site) endonuclease

GENE NAME

APEX1

SYNONYMS

APEN, AP endonuclease 1, APE-1, APEX1, APE, APE1, APEX, APX, HAP1, REF1

SEQUENCE SIMILARITIES

Belongs to the DNA repair enzymes AP/ExoA family.

POST-TRANSLATIONAL MODIFICATION

Phosphorylated. Phosphorylation by kinase PKC or casein kinase CK2 results in enhanced redox activity that stimulates binding of the FOS/JUN AP-1 complex to its cognate binding site. AP-endodeoxyribonuclease activity is not affected by CK2-mediated phosphorylation. Phosphorylation of Thr-233 by CDK5 reduces AP-endodeoxyribonuclease activity resulting in accumulation of DNA damage and contributing to neuronal death.; Acetylated on Lys-6 and Lys-7. Acetylation is increased by the transcriptional coactivator EP300 acetyltransferase, genotoxic agents like H(2)O(2) and methyl methanesulfonate (MMS). Acetylation increases its binding affinity to the negative calcium response element (nCaRE) DNA promoter. The acetylated form induces a stronger binding of YBX1 to the Y-box sequence in the MDR1 promoter than the unacetylated form. Deacetylated on lysines. Lys-6 and Lys-7 are deacetylated by SIRT1.; Cleaved at Lys-31 by granzyme A to create the mitochondrial form; leading in reduction of binding to DNA, AP endodeoxynuclease activity, redox activation of transcription factors and to enhanced cell death. Cleaved by granzyme K; leading to intracellular ROS accumulation and enhanced cell death after oxidative stress.; Cys-65 and Cys-93 are nitrosylated in response to nitric oxide (NO) and lead to the exposure of the nuclear export signal (NES).; Ubiquitinated by MDM2; leading to translocation to the cytoplasm and proteasomal degradation.

SUBCELLULAR LOCATION

Nucleus. Nucleus, nucleolus. Nucleus speckle. Endoplasmic reticulum. Cytoplasm. Note=Detected in the cytoplasm of B-cells stimulated to switch (By similarity). Colocalized with SIRT1 in the nucleus. Colocalized with YBX1 in nuclear speckles after genotoxic stress. Together with OGG1 is recruited to nuclear speckles in UVA-irradiated cells. Colocalized with nucleolin and NPM1 in the nucleolus. Its nucleolar localization is cell cycle dependent and requires active rRNA transcription. Colocalized with calreticulin in the endoplasmic reticulum. Translocation from the nucleus to the cytoplasm is stimulated in presence of nitric oxide (NO) and function in a CRM1-dependent manner, possibly as a consequence of demasking a nuclear export signal (amino acid position 64-80). S-nitrosylation at Cys-93 and Cys-310 regulates its nuclear-cytosolic shuttling. Ubiquitinated form is localized predominantly in the cytoplasm.; [DNA-(apurinic or apyrimidinic site) endonuclease, mitochondrial]: Mitochondrion. Note=The cleaved APEX2 is only detected in mitochondria (By similarity). Translocation from the cytoplasm to the mitochondria is mediated by ROS signaling and cleavage mediated by granzyme A. Tom20-dependent translocated mitochondrial APEX1 level is significantly increased after genotoxic stress.

FUNCTION

Multifunctional protein that plays a central role in the cellular response to oxidative stress. The two major activities of APEX1 are DNA repair and redox regulation of transcriptional factors. Functions as a apurinic/apyrimidinic (AP) endodeoxyribonuclease in the DNA base excision repair (BER) pathway of DNA lesions induced by oxidative and alkylating agents. Initiates repair of AP sites in DNA by catalyzing hydrolytic incision of the phosphodiester backbone immediately adjacent to the damage, generating a single-strand break with 5'-deoxyribose phosphate and 3'-hydroxyl ends. Does also incise at AP sites in the DNA strand of DNA/RNA hybrids, single-stranded DNA regions of R-loop structures, and single-stranded RNA molecules. Has a 3'-5' exoribonuclease activity on mismatched deoxyribonucleotides at the 3' termini of nicked or gapped DNA molecules during short-patch BER. Possesses a DNA 3' phosphodiesterase activity capable of removing lesions (such as phosphoglycolate) blocking the 3' side of DNA strand breaks. May also play a role in the epigenetic regulation of gene expression by participating in DNA demethylation. Acts as a loading factor for POLB onto non-incised AP sites in DNA and stimulates the 5'-terminal deoxyribose 5'-phosphate (dRp) excision activity of POLB. Plays a role in the protection from granzymes-mediated cellular repair leading to cell death. Also involved in the DNA cleavage step of class switch recombination (CSR). On the other hand, APEX1 also exerts reversible nuclear redox activity to regulate DNA binding affinity and transcriptional activity of transcriptional factors by controlling the redox status of their DNA-binding domain, such as the FOS/JUN AP-1 complex after exposure to IR. Involved in calcium-dependent down-regulation of parathyroid hormone (PTH) expression by binding to negative calcium response elements (nCaREs). Together with HNRNPL or the dimer XRCC5/XRCC6, associates with nCaRE, acting as an activator of transcriptional repression. Stimulates the YBX1-mediated MDR1 promoter activity, when acetylated at Lys-6 and Lys-7, leading to drug resistance. Acts also as an endoribonuclease involved in the control of single-stranded RNA metabolism. Plays a role in regulating MYC mRNA turnover by preferentially cleaving in between UA and CA dinucleotides of the MYC coding region determinant (CRD). In association with NMD1, plays a role in the rRNA quality control process during cell cycle progression. Associates, together with YBX1, on the MDR1 promoter. Together with NPM1, associates with rRNA. Binds DNA and RNA.