Recombinant Anti-PAK1 antibody [EPR20048] (ab223849)

All lanes : Anti-PAK1 antibody [EPR20048] (ab223849) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : PAK1 knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 61 kDa

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.

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.

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 

 

 

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.

All lanes : Anti-PAK1 antibody [EPR20048] (ab223849) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PAK1 CRISPR/Cas9 edited HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 61 kDa
Observed band size: 65 kDa why is the actual band size different from the predicted?

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 editedcell line ab264889 (CRISPR/Cas9 editedcell 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 : Anti-PAK1 antibody [EPR20048] (ab223849) at 1/1000 dilution

Lane 1 : SK-OV-3 (human ovarian cancer epithelial cell line) whole cell lysate
Lane 2 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate
Lane 3 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 4 : Human fetal brain lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lane 4 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution

Developed using the ECL technique.

Predicted band size: 61 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

 

Exposure time : Lane 1: 3 minutes; Lane 2: 30 seconds; Lane 3: 3 seconds; Lane 4: 15 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PAK1 antibody [EPR20048] (ab223849) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate
Lane 3 : SH-SY5Y (human euroblastoma cell line from bone marrow) whole cell lysate
Lane 4 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 61 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

 

Exposure time : Lane 1: 5 seconds; Lane 2: 3 seconds; Lane 3: 3 minutes; Lane 4: 1 minutes.

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PAK1 antibody [EPR20048] (ab223849) at 1/1000 dilution

Lane 1 : His-tagged human PAK1 (aa1-250) recombinant protein
Lane 2 : His-tagged human PAK2 (aa1-250) recombinant protein

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 61 kDa
Observed band size: 37 kDa why is the actual band size different from the predicted?

 

Exposure time : Lane 1: 1 minute; Lane 2: 3 minutes.

Blocking/Dilution buffer: 5% NFDM/TBST.

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.

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.