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    pak1-antibody-epr20048-ab223849.pdf

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Cell Biology Apoptosis Intracellular Kinases
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-PAK1 antibody [EPR20048] (ab223849)

  • Datasheet
  • SDS
  • Certificate of Compliance
Submit a review Submit a question References (7)

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Western blot - Anti-PAK1 antibody [EPR20048] (ab223849)
  • Immunocytochemistry/ Immunofluorescence - Anti-PAK1 antibody [EPR20048] (ab223849)
  • Flow Cytometry (Intracellular) - Anti-PAK1 antibody [EPR20048] (ab223849)
  • Immunoprecipitation - Anti-PAK1 antibody [EPR20048] (ab223849)
  • Western blot - Anti-PAK1 antibody [EPR20048] (ab223849)
  • Western blot - Anti-PAK1 antibody [EPR20048] (ab223849)
  • Western blot - Anti-PAK1 antibody [EPR20048] (ab223849)
  • Western blot - Anti-PAK1 antibody [EPR20048] (ab223849)
  • Immunocytochemistry/ Immunofluorescence - Anti-PAK1 antibody [EPR20048] (ab223849)
  • Flow Cytometry (Intracellular) - Anti-PAK1 antibody [EPR20048] (ab223849)
  • Anti-PAK1 antibody [EPR20048] (ab223849)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR20048] to PAK1
  • Suitable for: WB, ICC/IF, IP, Flow Cyt (Intra)
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

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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, IP, Flow Cyt (Intra)more details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: His-tagged human PAK1 (aa1-250) recombinant protein; HeLa, 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 (intra): SH-SY5Y and HeLa cells. IP: SH-SY5Y whole cell lysate.
  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

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

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.2
    Preservative: 0.1% Sodium azide
    Constituents: 0.05% BSA, 40% Glycerol (glycerin, glycerine), PBS
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR20048
  • Isotype

    IgG
  • Research areas

    • Cell Biology
    • Apoptosis
    • Intracellular
    • Kinases
    • Signal Transduction
    • Protein Phosphorylation
    • Ser / Thr Kinases
    • Other Kinases
    • Cancer
    • Signal transduction
    • Protein phosphorylation
    • Serine/threonine kinases
    • Other

Associated products

  • Alternative Versions

    • Anti-PAK1 antibody [EPR20048] - BSA and Azide free (ab242421)
  • Compatible Secondaries

    • VeriBlot for IP Detection Reagent (HRP) (ab131366)
    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
    • Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human PAK1 knockout HeLa cell line (ab264889)
  • KO cell lysates

    • Human PAK1 knockout HeLa cell lysate (ab257572)
  • Related Products

    • Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (ab195889)
    • Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204)

Applications

The Abpromise guarantee

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.
IP
1/30.
Flow Cyt (Intra)
1/50.
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.
IP
1/30.
Flow Cyt (Intra)
1/50.

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.
  • Target information above from: UniProt accession Q13153 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 5058 Human
    • Entrez Gene: 18479 Mouse
    • Entrez Gene: 29431 Rat
    • Omim: 602590 Human
    • SwissProt: Q13153 Human
    • SwissProt: O88643 Mouse
    • SwissProt: P35465 Rat
    • Unigene: 435714 Human
    • Unigene: 260227 Mouse
    • Unigene: 9149 Rat
    see all
  • Alternative names

    • 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

  • Western blot - Anti-PAK1 antibody [EPR20048] (ab223849)
    Western blot - 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.

  • Immunocytochemistry/ Immunofluorescence - Anti-PAK1 antibody [EPR20048] (ab223849)
    Immunocytochemistry/ Immunofluorescence - Anti-PAK1 antibody [EPR20048] (ab223849)

    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 Cytometry (Intracellular) - Anti-PAK1 antibody [EPR20048] (ab223849)
    Flow Cytometry (Intracellular) - Anti-PAK1 antibody [EPR20048] (ab223849)

    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 

     

     

  • Immunoprecipitation - Anti-PAK1 antibody [EPR20048] (ab223849)
    Immunoprecipitation - Anti-PAK1 antibody [EPR20048] (ab223849)

    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.

  • Western blot - Anti-PAK1 antibody [EPR20048] (ab223849)
    Western blot - Anti-PAK1 antibody [EPR20048] (ab223849)
    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.

  • Western blot - Anti-PAK1 antibody [EPR20048] (ab223849)
    Western blot - Anti-PAK1 antibody [EPR20048] (ab223849)
    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.

  • Western blot - Anti-PAK1 antibody [EPR20048] (ab223849)
    Western blot - Anti-PAK1 antibody [EPR20048] (ab223849)
    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.

  • Western blot - Anti-PAK1 antibody [EPR20048] (ab223849)
    Western blot - Anti-PAK1 antibody [EPR20048] (ab223849)
    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.

  • Immunocytochemistry/ Immunofluorescence - Anti-PAK1 antibody [EPR20048] (ab223849)
    Immunocytochemistry/ Immunofluorescence - Anti-PAK1 antibody [EPR20048] (ab223849)

    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 Cytometry (Intracellular) - Anti-PAK1 antibody [EPR20048] (ab223849)
    Flow Cytometry (Intracellular) - Anti-PAK1 antibody [EPR20048] (ab223849)

    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. 

     

     

  • Anti-PAK1 antibody [EPR20048] (ab223849)
    Anti-PAK1 antibody [EPR20048] (ab223849)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

Certificate of Compliance

To download a Certificate of Compliance, please enter your Lot number below:

References (7)

Publishing research using ab223849? Please let us know so that we can cite the reference in this datasheet.

ab223849 has been referenced in 7 publications.

  • Kuželová K  et al. Group I p21-activated kinases in leukemia cell adhesion to fibronectin. Cell Adh Migr 15:18-36 (2021). PubMed: 33464167
  • Li H  et al. Exosomes containing miR-451a is involved in the protective effect of cerebral ischemic preconditioning against cerebral ischemia and reperfusion injury. CNS Neurosci Ther 27:564-576 (2021). PubMed: 33533575
  • Chen YH  et al. Mesenchymal Stem Cell-Derived Exosomes Induced by IL-1ß Attenuate Urethral Stricture Through Let-7c/PAK1/NF-?B-Regulated Macrophage M2 Polarization. J Inflamm Res 14:3217-3229 (2021). PubMed: 34285545
  • Lin J  et al. MiR-23a induced the activation of CDC42/PAK1 pathway and cell cycle arrest in human cov434 cells by targeting FGD4. J Ovarian Res 13:90 (2020). PubMed: 32772928
  • Yao D  et al. Design, synthesis and biological evaluation of novel tetrahydrothieno [2,3-c]pyridine substitued benzoyl thiourea derivatives as PAK1 inhibitors in triple negative breast cancer. J Enzyme Inhib Med Chem 35:1524-1538 (2020). PubMed: 32752894
  • Grebenová D  et al. PAK1, PAK1?15, and PAK2: similarities, differences and mutual interactions. Sci Rep 9:17171 (2019). PubMed: 31748572
  • Liu C  et al. Epigenetically upregulated GEFT-derived invasion and metastasis of rhabdomyosarcoma via epithelial mesenchymal transition promoted by the Rac1/Cdc42-PAK signalling pathway. EBioMedicine 50:122-134 (2019). PubMed: 31761617

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