Overview

  • Product name
    Anti-PAK1 antibody [EPR20048]
    See all PAK1 primary antibodies
  • Description
    Rabbit monoclonal [EPR20048] to PAK1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ICC/IF, Flow Cyt, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human PAK1 aa 200-300. The exact sequence is proprietary.
    Database link: Q13153

  • Positive control
    • WB: His-tagged human PAK1 (aa1-250) recombinant protein; SK-OV-3, SH-SY5Y, HEK-293T, NIH/3T3 and PC-12 whole cell lysates; Human fetal brain lysate; Mouse and rat brain lysates. ICC/IF: SH-SY5Y and HeLa cells. Flow Cyt: SH-SY5Y and HeLa cells. IP: SH-SY5Y whole cell lysate.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab223849 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/1000. Detects a band of approximately 60 kDa (predicted molecular weight: 61 kDa).

In WB this antibody showed weak staining of PAK1 on HeLa cell lysate

ICC/IF 1/100.
Flow Cyt 1/50.
IP 1/30.

Target

  • Function
    The activated kinase acts on a variety of targets. Likely to be the GTPase effector that links the Rho-related GTPases to the JNK MAP kinase pathway. Activated by CDC42 and RAC1. Involved in dissolution of stress fibers and reorganization of focal complexes. Involved in regulation of microtubule biogenesis through phosphorylation of TBCB. Activity is inhibited in cells undergoing apoptosis, potentially due to binding of CDC2L1 and CDC2L2.
  • Sequence similarities
    Belongs to the protein kinase superfamily. STE Ser/Thr protein kinase family. STE20 subfamily.
    Contains 1 CRIB domain.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications
    Autophosphorylated when activated by CDC42/p21 and RAC1.
  • Cellular localization
    Cytoplasm. Cell junction > focal adhesion. Recruited to focal adhesions upon activation.
  • Information by UniProt
  • Database links
  • Alternative names
    • ADRB2 antibody
    • Alpha PAK antibody
    • Alpha-PAK antibody
    • MGC130000 antibody
    • MGC130001 antibody
    • p21 activated kinase 1 antibody
    • p21 protein (Cdc42/Rac) activated kinase 1 antibody
    • p21-activated kinase 1 antibody
    • p21/Cdc42/Rac1 activated kinase 1 (yeast Ste20 related) antibody
    • p21/Cdc42/Rac1-activated kinase 1 (STE20 homolog, yeast) antibody
    • p65 PAK antibody
    • p65-PAK antibody
    • P68-PAK antibody
    • PAK alpha antibody
    • PAK-1 antibody
    • Pak1 antibody
    • PAK1_HUMAN antibody
    • Paka antibody
    • PAKalpha antibody
    • Protein kinase MUK2 antibody
    • Rac/p21-activated kinase antibody
    • Serine/threonine-protein kinase PAK 1 antibody
    • STE20 homolog yeast antibody
    see all

Images

  • 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.

  • 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.

  • 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.

  • 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 : 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.

  • 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.

  • 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.

  • 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.

References

ab223849 has not yet been referenced specifically in any publications.

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