Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling CLASP1 (green) with ab108620 at 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 (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control: primary antibody (1/100) and secondary antibody, ab150120 Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

All lanes : Anti-CLASP1 antibody [EPR3409] (ab108620) at 1/10000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : CLASP1 knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : Jurkat whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 169 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab108620 observed at 169 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab108620 was shown to recognize CLASP1 in wild-type HAP1 cells as signal was lost at the expected MW in CLASP1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CLASP1 knockout samples were subjected to SDS-PAGE. Ab108620 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-CLASP1 antibody [EPR3409] (ab108620) at 1/20000 dilution (purified)

Lane 1 : U87-MG cell lysate
Lane 2 : HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 169 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Anti-CLASP1 antibody [EPR3409] (ab108620) at 1/50000 dilution (purified) + Rat brain tissue lysate at 10 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 169 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

All lanes : Anti-CLASP1 antibody [EPR3409] (ab108620) at 1/10000 dilution (unpurified)

Lane 1 : T47-D cell lysate
Lane 2 : C6 cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 169 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebral cortex tissue labelling CLASP1 with purified ab108620 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue labeling CLASP1 with purified ab108620 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling CLASP1 with unpurified ab108620 at 1/250.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling CLASP1 with unpurified ab108620 at 1/100.

Flow cytometry analysis of HeLa cells labelling CLASP1 with purified ab108620 at 1/50 (red). Cells were fixed with 80% methanol. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

Overlay histogram showing HeLa cells stained with unpurified ab108620 (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 (ab108620, 1/1000 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) (0.1μg/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.