Anti-PTPN12 antibody [EPR25171-63]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PTPN12 antibody [EPR25171-63] (AB289859)

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilised THP-1 (human monocytic leukemia monocyte cell line) cells labelling PTPN12 with ab289859 at 1/500 dilution (Red) compared with a Rabbit IgG, monoclonal - Isotype Control (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) (ab150081) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PTPN12 antibody [EPR25171-63] (AB289859)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized THP-1 (human monocytic leukemia monocyte) cells labeling PTPN12 with ab289859 at 1/200 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed secondary antibody at 1/1000 dilution (Green). Confocal image showing membranous and cytoplasmic staining in THP-1 cell line. The nuclear counterstain is DAPI (Blue). Tubulin is labeled using ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (Red).

Secondary antibody only control : Used PBS instead of primary antibody, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed secondary antibody at 1/1000 dilution.

• IP

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Immunoprecipitation - Anti-PTPN12 antibody [EPR25171-63] (AB289859)

PTPN12 was immunoprecipitated from THP-1 (human monocytic leukemia monocyte) whole cell lysate with ab289859 at 1/30 dilution (2 μg in 0.35 mg lysate). VeriBlot for IP secondary antibody(HRP) (ab131366) was used as secondary antibody at 1/5000 dilution.

Lane 1 : THP-1 (human monocytic leukemia monocyte), whole cell lysate 10 μg (Input)

Lane 2 : ab289859 IP in THP-1 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab289859 in THP-1 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST
Exposure time : 5 seconds

All lanes:

Immunoprecipitation - Anti-PTPN12 antibody [EPR25171-63] (ab289859)

Predicted band size: 88 kDa

Observed band size: 110-125 kDa

false

Exposure time: 5s

• WB

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Western blot - Anti-PTPN12 antibody [EPR25171-63] (AB289859)

5% NFDM/TBST was used as a blocking and diluting buffer.

Exposure times :

Lanes 1 - 2 : 125 seconds
Lane 3 : 92 seconds
Lanes 4 - 6 : 3 minutes

Lysates were made fresh and used immediately to minimize protein degradation.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 10625692).

All lanes:

Western blot - Anti-PTPN12 antibody [EPR25171-63] (ab289859) at 1/1000 dilution

Lane 1:

U937 (human histiocytic lymphoma monocyte), whole cell lysate at 20 µg

Lane 2:

HeLa (human epithelial cell line from cervix adenocarcinoma), whole cell lysate at 20 µg

Lane 3:

THP-1 (human monocytic leukemia monocyte), whole cell lysate at 20 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 20 µg

Lane 5:

C6 (rat glial tumor glial cell), whole cell lysate at 20 µg

Lane 6:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 20 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: 88 kDa

Observed band size: 110-125 kDa

false

• WB

Supplier Data

Western blot - Anti-PTPN12 antibody [EPR25171-63] (AB289859)

Blocking and diluting buffer : 5% NFDM/TBST.

ab181602 was used as a loading control at 1/1000000 dilution.

All lanes:

Western blot - Anti-PTPN12 antibody [EPR25171-63] (ab289859) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia monocyte) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

THP-1 transfected with siRNA specifically targeting PTPN12 whole cell lysate at 20 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 1/20000 dilution

Predicted band size: 88 kDa

Observed band size: 110 - 125 kDa

false

Exposure time: 20s