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

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.

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.

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.

Anti-Profilin 1 antibody [EPR6304] (ab124904) at 1/10000 dilution (Purified) + Human fetal brain lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 15 kDa
Observed band size: 12 kDa why is the actual band size different from the predicted?

All lanes : Anti-Profilin 1 antibody [EPR6304] (ab124904) at 1/10000 dilution (Purified)

Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates
Lane 2 : Mouse brain lysates
Lane 3 : Rat brain 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: 15 kDa
Observed band size: 12 kDa why is the actual band size different from the predicted?

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).

All lanes : Anti-Profilin 1 antibody [EPR6304] (ab124904) at 1/10000 dilution (Unpurified)

Lane 1 : Jurkat cell lysate
Lane 2 : JAR cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : 293T cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 15 kDa
Observed band size: 12 kDa why is the actual band size different from the predicted?

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

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

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

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.

Equilibrium disassociation constant (K_D)
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