重组Anti-MTAP抗体[EPR22570-76]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MTAP antibody [EPR22570-76] (AB254265)

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling MTAP with ab254265 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in human breast (PMID : 26751376, 18712977) 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.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MTAP antibody [EPR22570-76] (AB254265)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A549 (human lung carcinoma cell line) and HT-29 (human colorectal adenocarcinoma cell line) cells labeling MTAP with ab254265 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and nuclear staining in HT-29 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 (red).

SEcondary antibody only control : Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Negative control : A549（PMID : 8971171.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MTAP antibody [EPR22570-76] (AB254265)

Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling MTAP with ab254265 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in stroma cells and no staining in tumor cells of human bladder cancer (PMID : 27270441) 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.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MTAP antibody [EPR22570-76] (AB254265)

Intracellular flow cytometric analysis of4% paraformaldehyde-fixed, 90% methanol-permeabilized A549 (human lung carcinoma cell line) (Left) anf H-29 (human colorectal adenocarcinoma cell line) (Right) cells labeling MTAP with ab254265 at 1/400 dilution (red) compared with Recombinant Rabbit IgG, monoclonal [EPR25A] - 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) (ab150077), at 1/2000 dilution was used as the secondary antibody.

Negative control : A549 (PMID : 8971171.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MTAP antibody [EPR22570-76] (AB254265)

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling MTAP with ab254265 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in mouse liver (PMID : 15492751) 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.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MTAP antibody [EPR22570-76] (AB254265)

Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling MTAP with ab254265 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in rat liver (PMID : 15492751) 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.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• WB

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Western blot - Anti-MTAP antibody [EPR22570-76] (AB254265)

Blocking and dilution buffer : 5% NFDM/TBST.

Negative control : A549 (PMID : 17548352).

The expression molecular weight observed is consistent with what has been described in the literature (PMID : 15492751).

Exposure times.

Lanes 1-2 : 10 seconds; Lane3 : 5.5 seconds; Lanes 4-8 : 3.25 seconds.

All lanes:

Western blot - Anti-MTAP antibody [EPR22570-76] (ab254265) at 1/1000 dilution

Lane 1:

Human liver lysate at 20 µg

Lane 2:

Human placenta lysate at 20 µg

Lane 3:

HT-29 (human colorectal adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 4:

HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Lane 5:

HeLa (human epithelial cell line from cervix adenocarcinoma0 whole cell lysate at 20 µg

Lane 6:

Raji 9human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 7:

Caco-2 (human colorectal adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 8:

A549 9human colorectal adenocarcinoma cell line) whole cell lysate at 20 µg

Secondary

Lanes 1 - 2:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Lanes 3 - 8:

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

Predicted band size: 31 kDa

Observed band size: 32 kDa

false

• WB

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Western blot - Anti-MTAP antibody [EPR22570-76] (AB254265)

anes 1- 4 : Merged signal (red and green). Green - ab126770 observed at 32 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab126770 Anti-MTAP antibody [EPR6893] was shown to specifically react with MTAP in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265272 (knockout cell lysate ab257194) was used. Wild-type and MTAP knockout samples were subjected to SDS-PAGE. ab126770 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-MTAP antibody [EPR22570-76] (ab254265) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

MTAP knockout HeLa cell lysate at 20 µg

Lane 3:

HT-29 cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Predicted band size: 31 kDa

Observed band size: 32 kDa

false

• WB

Lab

Western blot - Anti-MTAP antibody [EPR22570-76] (AB254265)

Lanes 1-4 : Merged signal (red and green). Green - ab254265 observed at 32 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab254265 Anti-MTAP antibody [EPR22570-76] was shown to specifically react with MTAP in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265272 (knockout cell lysate ab257194) was used. Wild-type and MTAP knockout samples were subjected to SDS-PAGE. ab254265 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-MTAP antibody [EPR22570-76] (ab254265) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

MTAP knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human MTAP knockout HeLa cell line (<a href='/products/cell-lines/human-mtap-knockout-hela-cell-line-ab265272'>ab265272</a>)

Lane 3:

HT-29 cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Predicted band size: 31 kDa

Observed band size: 32 kDa

false

• WB

Unknown

Western blot - Anti-MTAP antibody [EPR22570-76] (AB254265)

Lanes 1-4 : Merged signal (red and green). Green - ab254265 observed at 32 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab254265 Anti-MTAP antibody [EPR22570-76] was shown to specifically react with MTAP in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265272 (knockout cell lysate ab257194) was used. Wild-type and MTAP knockout samples were subjected to SDS-PAGE. ab254265 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-MTAP antibody [EPR22570-76] (ab254265) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

MTAP knockout HeLa cell lysate at 20 µg

Lane 3:

HT-29 cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Predicted band size: 31 kDa

Observed band size: 32 kDa

false

• WB

Unknown

Western blot - Anti-MTAP antibody [EPR22570-76] (AB254265)

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure times :
Lanes 1-4 : 15 seconds; Lane 5 : 10 seconds; Lane 6 : 3.25 seconds.

The expression molecular weight observed is consistent with what has been described in the literature (PMID : 15492751).

All lanes:

Western blot - Anti-MTAP antibody [EPR22570-76] (ab254265) at 1/1000 dilution

Lane 1:

Mouse skin lysate at 20 µg

Lane 2:

Mouse liver lysate at 20 µg

Lane 3:

Rat skin lysate at 20 µg

Lane 4:

Rat liver lysate at 20 µg

Lane 5:

RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 20 µg

Lane 6:

NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 20 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 1/100000 dilution

Predicted band size: 31 kDa

Observed band size: 32 kDa

false