All lanes : Anti-MTAP antibody [EPR6893] (ab126770) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : MTAP knockout HeLa cell lysate
Lane 3 : HT-29 cell lysate
Lane 4 : A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 31 kDa
Observed band size: 32 kDa why is the actual band size different from the predicted?

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 : Anti-MTAP antibody [EPR6893] (ab126770) at 1/10000 dilution (purified)

Lane 1 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
Lane 2 : C6 (Rat glial tumor glial cell) whole cell lysates
Lane 3 : Mouse kidney lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 31 kDa



Blocking and diluting buffer : 5% NFDM/TBST

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.

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.

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.

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.

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).

All lanes : Anti-MTAP antibody [EPR6893] (ab126770) at 1/2000 dilution (purified)

Lane 1 : HT-29 (Human colorectal adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 31 kDa



Blocking and diluting buffer : 5% NFDM/TBST

All lanes : Anti-MTAP antibody [EPR6893] (ab126770) at 1/1000 dilution (unpurified)

Lane 1 : 293T cell lysates
Lane 2 : HT29 cell lysates
Lane 3 : C6 cell lysates
Lane 4 : RAW 264.7 cell lysates
Lane 5 : NIH 3T3 cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-Rabbit HRP at 1/2000 dilution

Predicted band size: 31 kDa

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.

Equilibrium disassociation constant (K_D)
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