Anti-MTAP antibody [EPR6893]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MTAP antibody [EPR6893] (AB126770)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling MTAP with purified ab126770 at 1/90 dilution (10 μg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MTAP antibody [EPR6893] (AB126770)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labeling MTAP with Purified ab126770 at 1/250 dilution. Cells were fixed with 4% PFA and permeabilized with 0.1% tritonX-100. ab150077 Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI. PBS was used instead of the primary antibody as the negative control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MTAP antibody [EPR6893] (AB126770)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human lung carcinoma tissue sections labeling MTAP with Purified ab126770 at 1 : 1000 dilution (0.89 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1 : 0 dilution. PBS instead of the primary antibody was used as the negative control.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MTAP antibody [EPR6893] (AB126770)

Overlay histogram showing HeLa cells stained with unpurified ab126770 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab126770, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MTAP antibody [EPR6893] (AB126770)

Formalin-fixed, paraffin-embedded human kidney tissue stained for MTAP with unpurified ab126770 (1/50 dilution) in immunohistochemical analysis.

Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

• WB

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Western blot - Anti-MTAP antibody [EPR6893] (AB126770)

All lanes:

Western blot - Anti-MTAP antibody [EPR6893] (ab126770) at 1/1000 dilution

Lane 1:

293T cell lysates at 10 µg

Lane 2:

HT29 cell lysates at 10 µg

Lane 3:

C6 cell lysates at 10 µg

Lane 4:

RAW 264.7 cell lysates at 10 µg

Lane 5:

NIH 3T3 cell lysates at 10 µg

Secondary

All lanes:

Goat anti-Rabbit HRP at 1/2000 dilution

Predicted band size: 31 kDa

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• WB

Lab

Western blot - Anti-MTAP antibody [EPR6893] (AB126770)

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 [EPR6893] (ab126770) 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='/en-us/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

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• WB

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Western blot - Anti-MTAP antibody [EPR6893] (AB126770)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-MTAP antibody [EPR6893] (ab126770) at 1/10000 dilution

Lane 1:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 15 µg

Lane 2:

C6 (Rat glial tumor glial cell) whole cell lysates at 15 µg

Lane 3:

Mouse kidney lysates at 15 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: 31 kDa

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• IP

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Immunoprecipitation - Anti-MTAP antibody [EPR6893] (AB126770)

ab126770 (purified) at 1 : 50 dilution (2μg) immunoprecipitating MTAP in HT-29 whole cell lysate.
Lane 1 (input) : HT-29 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+) : ab126770 & HT-29 whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab126770 in HT-29 whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-MTAP antibody [EPR6893] (ab126770)

Predicted band size: 31 kDa

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• WB

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Western blot - Anti-MTAP antibody [EPR6893] (AB126770)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-MTAP antibody [EPR6893] (ab126770) at 1/2000 dilution

Lane 1:

HT-29 (Human colorectal adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates 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: 31 kDa,71 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-MTAP antibody [EPR6893] (AB126770)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.