All lanes : Anti-Profilin 1 antibody [EPR6304] (ab124904) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : PFN1 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: 15 kDa

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124904).

Lanes 1 - 4: Merged signal (red and green). Green - ab124904 observed at 15 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab124904 was shown to specifically react with in wild-type HAP1 cells as signal was lost in PFN1 knockout cells. Wild-type and PFN1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% NF Milk. Ab124904 and ab8245 (Mouse anti-GAPDH 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.

This image was made using ab124904 which is the same antibody as ab232020 with BSA and Azide
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast carcinoma tissue sections labeling Profilin 1 with Purified ab124904 at 1:800 dilution (0.18 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

This image was made using ab124904 which is the same antibody as ab232020 with BSA and Azide
Immunocytochemistry/ Immunofluorescence analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling Profilin 1 with Purified ab124904 at 1:100 dilution (1.4 µg/ml). Cells were fixed in 100% Methanol. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

This image was made using ab124904 which is the same antibody as ab232020 with BSA and Azide
Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Profilin 1 with Purified ab124904 at 1:200 dilution (1 µg/ml) (red). Cells were fixed with 80% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Overlay histogram showing HeLa cells stained with unpurified ab124904 (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 (ab124904, 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124904).

Unpurified ab124904, at 1/250 dilution, staining Profilin 1 in HeLa cells by Immunofluorescence.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124904).

Equilibrium disassociation constant (K_D)
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This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124904).

Unpurified ab124904, at 1/500 dilution, staining Profilin 1 in paraffin-embedded human colon tissue by Immunohistochemistry.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124904).

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