Anti-Profilin 1 antibody [EPR6304]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Profilin 1 antibody [EPR6304] (AB124904)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Profilin 1 antibody [EPR6304] (AB124904)

Overlay histogram showing HeLa cells stained with unpurifiedab124904 (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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Profilin 1 antibody [EPR6304] (AB124904)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Profilin 1 antibody [EPR6304] (AB124904)

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Profilin 1 antibody [EPR6304] (AB124904)

Intracellular 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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Profilin 1 antibody [EPR6304] (AB124904)

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.

• WB

Lab

Western blot - Anti-Profilin 1 antibody [EPR6304] (AB124904)

ab124904 was shown to react with PFN1 in wild-type HAP1 cells in Western blot with loss of signal observed in a PFN1 knockout cell line. Wild-type HAP1 and PFN1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab124904 overnight at 4 °C at a 1/30000 dilution. Blots were incubated with secondary antibodies at 0.2ug/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-Profilin 1 antibody [EPR6304] (ab124904) at 1/30000 dilution

Lane 1:

Wild-type HAP1 lysate at 20 µg

Lane 2:

PFN1 knock-out HAP1 lysate at 20 µg

Observed band size: 15 kDa

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• WB

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Western blot - Anti-Profilin 1 antibody [EPR6304] (AB124904)

All lanes:

Western blot - Anti-Profilin 1 antibody [EPR6304] (ab124904) at 1/10000 dilution

Lane 1:

Jurkat cell lysate at 10 µg

Lane 2:

JAR cell lysate at 10 µg

Lane 3:

HepG2 cell lysate at 10 µg

Lane 4:

293T cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 15 kDa

Observed band size: 12 kDa

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• WB

Lab

Western blot - Anti-Profilin 1 antibody [EPR6304] (AB124904)

All lanes:

Western blot - Anti-Profilin 1 antibody [EPR6304] (ab124904) at 1/10000 dilution

Lane 1:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates at 15 µg

Lane 2:

Mouse brain lysates at 15 µg

Lane 3:

Rat brain lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 15 kDa

Observed band size: 12 kDa

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• WB

Lab

Western blot - Anti-Profilin 1 antibody [EPR6304] (AB124904)

All lanes:

Western blot - Anti-Profilin 1 antibody [EPR6304] (ab124904) at 1/10000 dilution

All lanes:

Human fetal brain lysates at 15 µg

Secondary

All lanes:

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

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• WB

Lab

Western blot - Anti-Profilin 1 antibody [EPR6304] (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.

All lanes:

Western blot - Anti-Profilin 1 antibody [EPR6304] (ab124904) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

PFN1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

Jurkat whole cell lysate at 20 µg

Predicted band size: 15 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-Profilin 1 antibody [EPR6304] (AB124904)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.