Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-PAK2 antibody [EP796Y] - BSA and Azide free (ab227990)

Overview

  • Product name
    Anti-PAK2 antibody [EP796Y] - BSA and Azide free
    See all PAK2 primary antibodies
  • Description
    Rabbit monoclonal [EP796Y] to PAK2 - BSA and Azide free
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IHC-P, Flow Cyt, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide (N terminal).

  • Positive control
    • WB: HeLa cytoplasmic lysate IHC-P; Human breast carcinoma tissue IF/ICC: T47D cell line.
  • General notes

    The formulation and the concentration of this product is compatible for metal-conjugation for mass cytometry (CyTOF®). 

    ab227990 is a PBS only buffer version of ab76293, containing no BSA or sodium azide, ideal for antibody labeling. Please refer to ab76293 for information on protocols, dilutions, and image data.

    Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

Properties

Applications

Our Abpromise guarantee covers the use of ab227990 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 Use at an assay dependent concentration. Predicted molecular weight: 58 kDa.
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

This antibody may not be suitable for IHC with mouse or rat samples
Use of HRP conjugated or polymerized HRP secondary antibody is recommended. Stronger signals have been found using the polymerized HRP secondary.

Flow Cyt Use at an assay dependent concentration.

ab199376 - Rabbit monoclonal IgG (Low endotoxin, Azide free), is suitable for use as an isotype control with this antibody.

ICC/IF Use at an assay dependent concentration.

Target

  • Function
    The activated kinase acts on a variety of targets. Phosphorylates ribosomal protein S6, histone H4 and myelin basic protein. Full length PAK 2 stimulates cell survival and cell growth. The process is, at least in part, mediated by phosphorylation and inhibition of pro-apoptotic BAD. Caspase-activated PAK-2p34 is involved in cell death response, probably involving the JNK signaling pathway. Cleaved PAK-2p34 seems to have a higher activity than the CDC42-activated form.
  • Tissue specificity
    Ubiquitously expressed. Higher levels seen in skeletal muscle, ovary, thymus and spleen.
  • 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
    Full length PAK 2 is autophosphorylated when activated by CDC42/p21. Following cleavage, both peptides, PAK-2p27 and PAK-2p34, become highly autophosphorylated, with PAK-2p27 being phosphorylated on serine and PAK-2p34 on threonine residues, respectively. Autophosphorylation of PAK-2p27 can occur in the absence of any effectors and is dependent on phosphorylation of Thr-402, because PAK-2p27 is acting as an exogenous substrate.
    During apoptosis proteolytically cleaved by caspase-3 or caspase-3-like proteases to yield active PAK-2p34.
    Ubiquitinated, leading to its proteasomal degradation.
    PAK-2p34 is myristoylated.
  • Cellular localization
    Cytoplasm and Nucleus. Cytoplasm > perinuclear region. Membrane. Interaction with ARHGAP10 probably changes PAK-2p34 location to cytoplasmic perinuclear region. Myristoylation changes PAK-2p34 location to the membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • C-t-PAK2 antibody
    • CB422 antibody
    • EC 2.7.11.1 antibody
    • Gamma PAK antibody
    • Gamma-PAK antibody
    • hPAK65 antibody
    • Kinase antibody
    • p21 (CDKN1A) activated kinase 2 antibody
    • p21 (CDKN1A)-activated kinase 2a antibody
    • p21 activated kinase 2 antibody
    • p21 protein (Cdc42/Rac)-activated kinase 2 antibody
    • p21 protein Cdc42 Rac activated kinase 2 antibody
    • p21-activated kinase 2 antibody
    • p21-activated kinase, 65-KD antibody
    • p21-activated protein kinase I antibody
    • p21CDKN1A activated kinase 2 antibody
    • p27 antibody
    • p34 antibody
    • p58 antibody
    • p65PAK antibody
    • PAK 2 antibody
    • PAK-2 antibody
    • PAK-2p34 antibody
    • Pak2 antibody
    • PAK2_HUMAN antibody
    • PAK65 antibody
    • PAKgamma antibody
    • S6 H4 kinase antibody
    • S6/H4 kinase antibody
    • Serine threonine protein kinase PAK 2 antibody
    • Serine/threonine protein kinase PAK 2 antibody
    see all

Images

  • This WB data was generated using the same anti-PAK2 antibody clone, EP796Y, in a different buffer formulation (cat# ab76293).

    Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: PAK2 knockout HAP1 cell lysate (20 µg) 
    Lane 3: HeLa cell lysate (20 µg)
    Lane 4: Human lymph node tissue lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab76293 observed at 60 kDa. Red - loading control, ab8245, observed at 37 kDa.

    Unpurified ab76293 was shown to specifically react with PAK2 when PAK2 knockout samples were used. Wild-type and PAK2 knockout samples were subjected to SDS-PAGE. ab76293 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 respectively and incubated overnight at 4°C. 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 1/10000 dilution for 1 h at room temperature before imaging.

  • Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling PAK2 with purified ab76293 at 1:20 dilution (10 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilized with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). 

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76293).

  • Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling PAK2 with purified ab76293 at 1:100 dilution (2.0μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76293).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue sections labeling PAK2 with Purified ab76293 at 1:100 dilution (2.02 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76293).

  • ICC/IF image of unpurified ab76293 stained T47D cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab76293, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76293).

  • Overlay histogram showing HeLa cells stained with unpurified ab76293 (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 (ab76293, 1/100 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) (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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab76293).

  • This IHC data was generated using the same anti-PAK2 antibody clone, EP796Y, in a different buffer formulation (cat# ab76293).

    Unpurified ab76293, at a 1/100 dilution, staining PAK2 in paraffin embedded human breast carcinoma tissue by Immunohistochemistry.

References

This product has been referenced in:
  • Jiang N  et al. In vivo quantitative phosphoproteomic profiling identifies novel regulators of castration-resistant prostate cancer growth. Oncogene : (2014). Mouse . Read more (PubMed: 25065596) »
  • Chen G  et al. TGFß receptor I transactivation mediates stretch-induced Pak1 activation and CTGF upregulation in mesangial cells. J Cell Sci 126:3697-712 (2013). Read more (PubMed: 23781022) »
See all 3 Publications for this product

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