Immunohistochemical analysis of paraffin-embedded human colon cancer tissue labeling PTP1B with ab244207 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody. Cytoplasmic staining in human colon cancer (PMID:27752061) is observed. Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

All lanes : Anti-PTP1B antibody [EPR22474] (ab244207) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PTPN1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 50 kDa
Observed band size: 50 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab244207 observed at 50 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab244207 was shown to react with PTP1B in wild-type HeLa cells in western blot. The band observed in knockout cell line ab265014 (knockout cell lysate ab257617) lane below 50kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and PTPN1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab244207 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated 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) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labeling PTP1B with ab244207 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in HeLa cells is observed. The nuclear counterstain is DAPI (blue).
Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red).
The negative control is the secondary antibody only.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cell line labeling PTP1B with ab244207 at 1/500 (red) compared with a Rabbit monoclonal IgG (ab172730)isotype control (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

PTP1B  was immunoprecipitated from 0.35 mg HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate with ab244207 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab244207 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.
Lane 1: HCT 116 whole cell lysate 10 µg (Input).
Lane 2: ab244207 IP in HCT 116 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab244207 in HCT 116 whole cell lysate.
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 30 seconds.

The observed MW is consistent with what described in the literatures. (PMID: 18253097; PMID: 11895943; PMID: 19797268).

All lanes : Anti-PTP1B antibody [EPR22474] (ab244207) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : PTP1B knockout HAP1 whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 50 kDa

ab244207 was shown to specifically react with PTP1B in wild-type HAP1 cells as signal was lost in PTP1B knockout cells. Wild-type and PTP1B knockout samples were subjected to SDS-PAGE. ab244207 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique.

The expression profile observed is consistent with what has been described in the literature (PMID: 18253097; PMID: 11895943; PMID: 19797268). The bands below 50 kDa may represent truncated forms and cleaved fragments.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

All lanes : Anti-PTP1B antibody [EPR22474] (ab244207) at 1/1000 dilution

Lane 1 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 2 : A549 (human lung carcinoma epithelial cell) whole cell lysate
Lane 3 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 50 kDa

The expression profile observed is consistent with what has been described in the literature (PMID: 18253097; PMID: 11895943; PMID: 19797268). The bands below 50 kDa may represent truncated forms and cleaved fragments.

Blocking/Dilution buffer: NFDM/TBST.

Exposure time: 37 seconds.

All lanes : Anti-PTP1B antibody [EPR22474] (ab244207) at 1/1000 dilution

Lane 1 : RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 2 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 50 kDa

The expression profile observed is consistent with what has been described in the literature (PMID: 18253097; PMID: 11895943; PMID: 19797268). The bands below 50 kDa may represent truncated forms and cleaved fragments.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: 48 seconds.

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling PTP1B with ab244207 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Higher cytoplasmic expression in human breast cancer than that of adjacent normal tissues (PMID: 27465552) is observed. Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HCT 116 (human colorectal carcinoma epithelial cell) cells labeling PTP1B with ab244207 at 1/50 dilution, followed by a Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in HCT 116 cells. The nuclear counterstain is DAPI (blue).
Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red).
The negative control is the secondary antibody only.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Wild type and PTP1B-knockout HAP1 (Human chronic myelogenous leukemia near-haploid cell line) cells labeling PTP1B with ab244207 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing no staining in PTP1B-knockout HAP1 cells. The nuclear counterstain is DAPI (blue).
Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red).
The negative control is the secondary antibody only.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HCT 116 (human colorectal carcinoma epithelial cell) cell line labeling PTP1B with ab244207 at 1/50 (red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.