重组Anti-Met (c-Met) (phospho Y1349)抗体[EP2367Y]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Met (c-Met) (phospho Y1349) antibody [EP2367Y] (AB68141)

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using ab68141 at a dilution of 1/50.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

• IP

Lab

Immunoprecipitation - Anti-Met (c-Met) (phospho Y1349) antibody [EP2367Y] (AB68141)

ab68141 at 1/150 dilution immunoprecipitating Met (c-Met) (phospho Y1349) in HeLa (human cervix adenocarcinoma) whole cell lysate observed at 150 kDa (lanes 1 and 2).

Lane 1 (input) : HeLa cells starved for 24 hours, then treated with 40 ng/mL HGF for 5 minutes whole cell lysate, 10μg
Lane 2 (+) : ab68141 + HeLa cells starved for 24 hours, then treated with 40 ng/mL HGF for 5 minutes whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab68141 in HeLa cells starved for 24 hours, then treated with 40 ng/mL HGF for 5 minutes whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking and Diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Met (c-Met) (phospho Y1349) antibody [EP2367Y] (ab68141)

Predicted band size: 155 kDa

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Exposure time: 3s

• WB

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Western blot - Anti-Met (c-Met) (phospho Y1349) antibody [EP2367Y] (AB68141)

All lanes:

Western blot - Anti-Met (c-Met) (phospho Y1349) antibody [EP2367Y] (ab68141) at 1/10000 dilution

Lane 1:

A431 cell lysate, untreated at 10 µg

Lane 2:

A431 cell lysate, treated with AP at 10 µg

Secondary

All lanes:

HRP-labeled goat anti-rabbit at 1/2000 dilution

Predicted band size: 155 kDa

Observed band size: ~150 kDa

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• WB

Lab

Western blot - Anti-Met (c-Met) (phospho Y1349) antibody [EP2367Y] (AB68141)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The molecular weight of Met (c-Met) (phospho Y1349) and Met (c-Met) is different. ab51067 could only recognize the pro-Met which is 190kDa, but ab68141 recognizes theβ-subunits which is 145kDa.

All lanes:

Western blot - Anti-Met (c-Met) (phospho Y1349) antibody [EP2367Y] (ab68141) at 1/5000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) serum starved for 24 h. Whole cell lysates at 15 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) serum starved for 24 h and then treated with hepatocyte growth factor at 40ng/ml for 5 min. Whole cell lysates at 15 µg

Lane 3:

HeLa (Human cervix adenocarcinoma epithelial cell) serum starved for 24 h and then treated with hepatocyte growth factor at 40ng/ml for 5 min. Whole cell lysates. Then the membrane was incub at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 155 kDa

Observed band size: 150 kDa

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Exposure time: 30s

• Dot

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Dot Blot - Anti-Met (c-Met) (phospho Y1349) antibody [EP2367Y] (AB68141)

Dot blot analysis of Met (c-Met) (phospho Y1349) phospho peptide (Lane 1) Met (c-Met) Non-phospho peptide (Lane 2) labelling Met (c-Met) (phospho Y1349) with ab68141 at a dilution of 1/1000. Peroxidase conjugated goat anti-rabbit IgG (H+L)) was used as the secondary antibody at a dilution of 1/2500.

Blocking and diluting buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.

• WB

CiteAb

Western blot - Anti-Met (c-Met) (phospho Y1349) antibody [EP2367Y] (AB68141)

Met (c-Met) (phospho Y1349) western blot using anti-Met (c-Met) (phospho Y1349) antibody [EP2367Y] ab68141. Publication image and figure legend from Zhao, Q., Zhu, H. P., et al., 2020, Int J Mol Sci, PubMed 32156008.

ab68141 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab68141 please see the product overview.

(A) Cells were incubated with various concentrations (0, 20, or 40 μg/mL) of DHP1808 for 24 h. Expression or phosphorylation levels of HSP90 client proteins were determined by western blot analysis; (B) Cells were incubated with various concentrations (0, 20, or 40 μg/mL) of DHP1808 for 24 h. Expression or phosphorylation levels of proteins associated with MAPK signaling pathway were determined by western blot analysis; (C) Cells were incubated with various concentrations (0, 20, or 40 μg/mL) of DHP1808 for 24 h. Expression or phosphorylation level of proteins associated with the PI3K-Akt signaling pathway was determined by western blot analysis; (D). The fold change of EGFR expression was determined by qPCR analysis.

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