Anti-PAK1 antibody [EPR20048] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PAK1 antibody [EPR20048] - BSA and Azide free (AB242421)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling PAK1 with ab223849 at 1/50 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab223849).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-PAK1 antibody [EPR20048] - BSA and Azide free (AB242421)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized SH-SY5Y (human neuroblastoma cell line from bone marrow) cell line labeling PAK1 with ab223849 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue).

Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab223849).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PAK1 antibody [EPR20048] - BSA and Azide free (AB242421)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized SH-SY5Y (human neuroblastoma cell line from bone marrow) cells labeling PAK1 with ab223849 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on SH-SY5Y cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab223849).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PAK1 antibody [EPR20048] - BSA and Azide free (AB242421)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling PAK1 with ab223849 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab223849).

• IP

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Immunoprecipitation - Anti-PAK1 antibody [EPR20048] - BSA and Azide free (AB242421)

PAK1 was immunoprecipitated from 0.35 mg SH-SY5Y (human neuroblastoma cell line from bone marrow) whole cell lysate with ab223849 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab223849 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : SH-SY5Y whole cell lysate 10 μg (Input).

Lane 2 : ab223849 IP in SH-SY5Y whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab223849 in SH-SY5Y whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 1 second.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab223849).

All lanes:

Immunoprecipitation - Anti-PAK1 antibody [EPR20048] (<a href='/en-us/products/primary-antibodies/pak1-antibody-epr20048-ab223849'>ab223849</a>)

Predicted band size: 61 kDa

false

• WB

Lab

Western blot - Anti-PAK1 antibody [EPR20048] - BSA and Azide free (AB242421)

This data was developed using the same antibody clone in a different buffer formulation (ab223849).

Lanes 1- 2 : Merged signal (red and green). Green - ab223849 observed at 65 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab223849 was shown to react with PAK1 in wild-type HeLa cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab264889 (CRISPR/Cas9 edited cell lysate ab257572) lane below 65kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and PAK1 CRISPR/Cas9 edited HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab223849 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PAK1 antibody [EPR20048] (<a href='/en-us/products/primary-antibodies/pak1-antibody-epr20048-ab223849'>ab223849</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PAK1 CRISPR/Cas9 edited HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human PAK1 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-pak1-knockout-hela-cell-line-ab264889'>ab264889</a>)

Predicted band size: 61 kDa

Observed band size: 65 kDa

false

• WB

Supplier Data

Western blot - Anti-PAK1 antibody [EPR20048] - BSA and Azide free (AB242421)

Lanes 1 - 2 : Merged signal (red and green). Green - ab223849 observed at 61 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab223849 was shown to specifically react with PAK1 in wild-type HAP1 cells as signal was lost in PAK1 knockout cells. Wild-type and PAK1 knockout samples were subjected to SDS-PAGE. ab223849 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 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.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

All lanes:

Western blot - Anti-PAK1 antibody [EPR20048] (<a href='/en-us/products/primary-antibodies/pak1-antibody-epr20048-ab223849'>ab223849</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

PAK1 knockout HAP1 whole cell lysate at 20 µg

Predicted band size: 61 kDa

false