Anti-APE1 antibody [EPR18378-45] - ChIP Grade

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-APE1 antibody [EPR18378-45] - ChIP Grade (AB189474)

Immunohistochemical analysis of paraffin-embedded human ovarian cancer tissue labeling APE1 with ab189474 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining on tumor cells of human ovarian carcinoma (PMID : 20087352) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-APE1 antibody [EPR18378-45] - ChIP Grade (AB189474)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HCT 116 (human colorectal carcinoma cell line) cells labeling APE1 with ab189474 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HCT 116 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution.

-ve control : PBS, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-APE1 antibody [EPR18378-45] - ChIP Grade (AB189474)

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling APE1 with ab189474 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Immunohistochemical analysis of paraffin-embedded human ovarian cancer tissue labeling APE1 with ab189474 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear staining on tumor cells of human ovarian carcinoma (PMID : 20087352) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ChIP

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ChIP - Anti-APE1 antibody [EPR18378-45] - ChIP Grade (AB189474)

Chromatin was prepared from HCT 116 cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin, 5μg of ab189474 (blue), and 20μl of Protein A/G sepharose beads slurry (10μl of sepharose A beads + 10μl of sepharose G beads). 5μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (SYBR green approach).

ChIP was performed according to the literature (PMID : 23874636).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-APE1 antibody [EPR18378-45] - ChIP Grade (AB189474)

Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling APE1 with ab189474 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Mainly nuclear staining on hepatocytes of rat liver (PMID : 10643898) is observed. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-APE1 antibody [EPR18378-45] - ChIP Grade (AB189474)

Intracellular flow cytometric analysis of4% pasraformaldehyde-fixed, 90% methanol-permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cell line labeling APE1 with ab189474 at 1/500 dilution (red) compared with a Isotype control details (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue).

Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-APE1 antibody [EPR18378-45] - ChIP Grade (AB189474)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cells labeling APE1 with ab189474 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on NIH/3T3 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution.

-ve control : PBS, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution

• WB

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Western blot - Anti-APE1 antibody [EPR18378-45] - ChIP Grade (AB189474)

Blocking and dilution buffer : 5% NFDM/TBST

Exposure times :

Lanes 1 & 2 : 1 second

Lane 3 : 5 seconds

Lane 4 : 3 seconds

All lanes:

Western blot - Anti-APE1 antibody [EPR18378-45] - ChIP Grade (ab189474) at 1/5000 dilution

Lane 1:

HCT 116 (human colorectal carcinoma cell line) whole cell lysate at 10 µg

Lane 2:

HeLa(human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 3:

NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 10 µg

Lane 4:

C6 (rat brain glioma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 35 kDa

Observed band size: 35 kDa

true

• WB

Supplier Data

Western blot - Anti-APE1 antibody [EPR18378-45] - ChIP Grade (AB189474)

Blocking and dilution buffer : 5% NFDM/TBST

Exposure times :

Lanes 1-3 : 1 second

Lane 4 : 4 seconds

All lanes:

Western blot - Anti-APE1 antibody [EPR18378-45] - ChIP Grade (ab189474) at 1/1000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Lane 4:

Human fetal spleen lysate at 10 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/2000 dilution

Predicted band size: 35 kDa

Observed band size: 35 kDa

true

• WB

Lab

Western blot - Anti-APE1 antibody [EPR18378-45] - ChIP Grade (AB189474)

Lanes 1 - 4 : Merged signal (red and green). Green - ab189474 observed at 37 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab189474 was shown to react with APEX1 in HAP1 wild-type cells in Western blot. Loss of signal was observed when APEX1 knockout sample was used. HAP1 wild-type and APEX1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween®) before incubation with ab189474 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-APE1 antibody [EPR18378-45] - ChIP Grade (ab189474) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

APEX1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HepG2 whole cell lysate at 20 µg

Lane 4:

HEK293 whole cell lysate at 20 µg

Predicted band size: 35 kDa

Observed band size: 37 kDa

false

• WB

Supplier Data

Western blot - Anti-APE1 antibody [EPR18378-45] - ChIP Grade (AB189474)

Blocking and dilution buffer : 5% NFDM/TBST

Exposure times :

Lanes 1-5 : 8 seconds

Lane 6 : 4 seconds

All lanes:

Western blot - Anti-APE1 antibody [EPR18378-45] - ChIP Grade (ab189474) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse heart lysate at 10 µg

Lane 3:

Mouse liver lysate at 10 µg

Lane 4:

Rat brain lysate at 10 µg

Lane 5:

Rat liver lysate at 10 µg

Lane 6:

Rat spleen lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 35 kDa

Observed band size: 35 kDa

true