All lanes : Anti-PTP1B antibody [EPR22468-6] (ab252928) at 1/2000 dilution

Lane 1 : HCT116 (human colorectal carcinoma epithelial cell), whole cell lysate
Lane 2 : Wild-type HAP1 whole cell lysate
Lane 3 : PTP1B knockout HAP1 whole cell lysate
Lane 4 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 5 : HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate
Lane 6 : TF-1 (human Erythroleukemia erythroblast), whole cell lysate
Lane 7 : A549 (human lung 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
Observed band size: 36,46,48,50 kDa why is the actual band size different from the predicted?

ab252928 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. ab252928 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/2000 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 50kDa may represent truncated forms and cleaved fragments.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lanes 1-3: 3 mins; Lanes 4-6: 15 secs; Lane 7: 3 mins.

Immunohistochemical analysis of paraffin-embedded human breast cancer (panel A) and its adjacent non-cancerous tissue (panel B) labeling PTP1B with ab252928 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Higher cytoplasmic staining in human breast cancer tissue (panel A) while weakly staining in its adjacent non-cancerous tissue (panel B) (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).

PTP1B was immunoprecipitated from 0.35 mg HCT116 (human colorectal carcinoma epithelial cell) whole cell lysate with ab252928 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab252928 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used at 1/5000 dilution.

Lane 1: HCT116 whole cell lysate 10 µg (Input).
Lane 2: ab252928 IP in HCT116 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab252928 in HCT116 whole cell lysate.

Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 3 seconds.

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

All lanes : Anti-PTP1B antibody [EPR22468-6] (ab252928) 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 - ab252928 observed at 50 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab252928 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. ab252928 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.

All lanes : Anti-PTP1B antibody [EPR22468-6] (ab252928) at 1/1000 dilution

Lane 1 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : SW480 (human colorectal adenocarcinoma epithelial cell), whole cell lysate
Lane 3 : Human placenta cell lysate
Lane 4 : Human fetal kidney cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lane 4 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 50 kDa
Observed band size: 36,46,50 kDa why is the actual band size different from the predicted?

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

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lanes 1-3: 92 secs; Lane 4: 3 mins.

All lanes : Anti-PTP1B antibody [EPR22468-6] (ab252928) at 1/1000 dilution

Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 2 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
Lane 3 : Mouse brain cell lysate
Lane 4 : Mouse spleen cell lysate
Lane 5 : Rat brain cell lysate
Lane 6 : Rat heart 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
Observed band size: 36,46,48,50 kDa why is the actual band size different from the predicted?

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

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lanes 1-2: 70 secs; Lane 3: 3 mins; Lane 4: 37 secs; Lanes 5-6: 3 mins.

Immunohistochemical analysis of paraffin-embedded human gastric cancer (panel A) and its adjacent non-cancerous tissue (panel B) labeling PTP1B with ab252928 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Higher cytoplasmic staining in human gastric cancer tissue (panel A) while weakly staining in its adjacent non-cancerous tissue (panel B) (PMID: 21442314) 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).

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling PTP1B with ab252928 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining in rat spleen (PMID:18716132) 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).

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling PTP1B with ab252928 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining in mouse spleen (PMID:18716132) 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).