Recombinant Anti-Mark3 antibody [EPR633Y] (ab52626)

ab52626 (purified) at a dilution of 1/20 immunoprecipitating Mark3 in K562 whole cell lysate.

Lane 1 (input): K562 whole cell lysate (10µg)

Lane 2 (+): ab52626 + K562 whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab52626 in K562 whole cell lysate.

For western blotting, ab131366 VeriBlot for IP (HRP) was used for detection (1/1000).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Lane 1: Wild-type HAP1 cell lysate (40 µg)
Lane 2: MARK3 knockout HAP1 cell lysate (40 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: K562 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab52626 observed at 85 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab52626 was shown to recognize Mark3 when Mark3 knockout samples were used, along with additional cross-reactive bands. Wild-type and Mark3 knockout samples were subjected to SDS-PAGE. Ab52626 and ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1:10,000 dilution respectively and incubated overnight at 4C. 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 1:10,000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Mark3 antibody [EPR633Y] (ab52626) at 1/2000 dilution (purified)

Lane 1 : K562 whole cell lysate
Lane 2 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Observed band size: 86 kDa why is the actual band size different from the predicted?



Blocking and dilution buffer: 5% NFDM/TBST

Anti-Mark3 antibody [EPR633Y] (ab52626) at 10 µg (purified) + NIH/3T3 whole cell lysate at 10 µg

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

Observed band size: 86 kDa why is the actual band size different from the predicted?



Blocking and dilution buffer: 5% NFDM/TBST

Immunocytochemistry/Immunofluorescence analysis of MCF-7 cells labelling Mark3 with purified ab52626 at a dilution of 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

Intracellular Flow Cytometry analysis ofHeLa cells labelling Mark3 with purified ab52626 at a dilution of 1/50 (red). Cells were fixed with 4% paraformaldehyde. An Alexa Fluorr^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies. 

 

 

Anti-Mark3 antibody [EPR633Y] (ab52626) at 1/2000 dilution (unpurified) + HeLa cell lysate at 10 µg

Secondary
HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Observed band size: 86 kDa why is the actual band size different from the predicted?

Overlay histogram showing HeLa cells stained with unpurified ab52626 (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 (unpurified ab52626, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluorr^® 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.