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  1. Link

    paxillin-antibody-e228-ab32115.pdf

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Signal Transduction Cytoskeleton / ECM Extracellular Matrix Structures Focal Adhesions
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Paxillin antibody [E228] (ab32115)

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Western blot - Anti-Paxillin antibody [E228] (ab32115)
  • Western blot - Anti-Paxillin antibody [E228] (ab32115)
  • Western blot - Anti-Paxillin antibody [E228] (ab32115)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Paxillin antibody [E228] (ab32115)
  • Immunocytochemistry/ Immunofluorescence - Anti-Paxillin antibody [E228] (ab32115)
  • Immunoprecipitation - Anti-Paxillin antibody [E228] (ab32115)
  • Flow Cytometry (Intracellular) - Anti-Paxillin antibody [E228] (ab32115)
  • Immunocytochemistry/ Immunofluorescence - Anti-Paxillin antibody [E228] (ab32115)
  • Western blot - Anti-Paxillin antibody [E228] (ab32115)
  • Immunocytochemistry/ Immunofluorescence - Anti-Paxillin antibody [E228] (ab32115)
  • Western blot - Anti-Paxillin antibody [E228] (ab32115)
  • ELISA - Anti-Paxillin antibody [E228] (ab32115)
  • Dot Blot - Anti-Paxillin antibody [E228] (ab32115)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Paxillin antibody [E228] (ab32115)
  • Anti-Paxillin antibody [E228] (ab32115)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [E228] to Paxillin
  • Suitable for: WB, ICC/IF, IHC-P, IP, Flow Cyt (Intra), Dot blot, ELISA
  • Knockout validated
  • Reacts with: Mouse, Rat, Human, Recombinant fragment

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Human PXN (Paxillin) knockout A-431 cell line (ab261892)

View more associated products

Overview

  • Product name

    Anti-Paxillin antibody [E228]
    See all Paxillin primary antibodies
  • Description

    Rabbit monoclonal [E228] to Paxillin
  • Host species

    Rabbit
  • Specificity

    The mouse and rat recommendation is based on the WB results. We do not guarantee IHC-P for mouse and rat.
  • Tested applications

    Suitable for: WB, ICC/IF, IHC-P, IP, Flow Cyt (Intra), Dot blot, ELISAmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human, Recombinant fragment
  • Immunogen

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

  • Positive control

    • WB: HeLa, NIH 3T3, HEK-293, C2C12, A431, PC-3 and Rat-1 whole cell lysates. ICC/IF: HeLa cells. IHC-P: Human colon and breast carcinoma. Flow Cyt (intra): HeLa cells. IP: HEK-293 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.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 40% Glycerol (glycerin, glycerine), PBS, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    E228
  • Isotype

    IgG
  • Research areas

    • Signal Transduction
    • Cytoskeleton / ECM
    • Extracellular Matrix
    • Structures
    • Focal Adhesions
    • Signal Transduction
    • Adapters
    • Cytoplasmic
    • Cancer
    • Signal transduction
    • Other

Associated products

  • Alternative Versions

    • Anti-Paxillin antibody [E228] - BSA and Azide free (ab238950)
  • 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 (HRP) (ab205718)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
  • Isotype control

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

    • Human PXN (Paxillin) knockout A-431 cell line (ab261892)
    • Human PXN (Paxillin) knockout HeLa cell line (ab264767)
  • KO cell lysates

    • Human PXN (Paxillin) knockout HeLa cell lysate (ab257044)
    • Human PXN (Paxillin) knockout A-431 cell lysate (ab261701)
  • KO cell pellets

    • Human PXN (Paxillin) knockout HeLa cell pellet (ab279009)
  • Positive Controls

    • NIH/3T3 whole cell lysate (ab7179)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab32115 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
1/10000. Predicted molecular weight: 64 kDa.
ICC/IF
1/100.

For unpurified use at 1/250

IHC-P
1/50. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

The mouse and rat recommendation is based on the WB results. We do not guarantee IHC-P for mouse and rat.

See IHC antigen retrieval protocols.

For unpurified use at 1/100 - 1/250

IP
1/20.

For unpurified use at 1/100

Flow Cyt (Intra)
1/20.
Dot blot
1/1000.
ELISA
Use at an assay dependent concentration.
Notes
WB
1/10000. Predicted molecular weight: 64 kDa.
ICC/IF
1/100.

For unpurified use at 1/250

IHC-P
1/50. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

The mouse and rat recommendation is based on the WB results. We do not guarantee IHC-P for mouse and rat.

See IHC antigen retrieval protocols.

For unpurified use at 1/100 - 1/250

IP
1/20.

For unpurified use at 1/100

Flow Cyt (Intra)
1/20.
Dot blot
1/1000.
ELISA
Use at an assay dependent concentration.

Target

  • Function

    Cytoskeletal protein involved in actin-membrane attachment at sites of cell adhesion to the extracellular matrix (focal adhesion).
  • Sequence similarities

    Belongs to the paxillin family.
    Contains 4 LIM zinc-binding domains.
  • Post-translational
    modifications

    Phosphorylated on tyrosine residues during integrin-mediated cell adhesion, embryonic development, fibroblast transformation and following stimulation of cells by mitogens.
  • Cellular localization

    Cytoplasm > cytoskeleton. Cell junction > focal adhesion.
  • Target information above from: UniProt accession P49023 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 5829 Human
    • Entrez Gene: 19303 Mouse
    • Entrez Gene: 360820 Rat
    • Omim: 602505 Human
    • SwissProt: P49023 Human
    • SwissProt: Q8VI36 Mouse
    • SwissProt: Q66H76 Rat
    • Unigene: 446336 Human
    • Unigene: 18714 Mouse
    • Unigene: 75 Rat
    see all
  • Alternative names

    • FLJ16691 antibody
    • FLJ23042 antibody
    • Paired box protein Pax 1 antibody
    • PAX 1 antibody
    • PAX1 antibody
    • PAXI_HUMAN antibody
    • Paxillin alpha antibody
    • Paxillin antibody
    • PXN antibody
    • PXN protein antibody
    see all

Images

  • Western blot - Anti-Paxillin antibody [E228] (ab32115)
    Western blot - Anti-Paxillin antibody [E228] (ab32115)
    All lanes : Anti-Paxillin antibody [E228] (ab32115) at 1/10000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : PXN knockout HeLa cell lysate
    Lane 3 : HEK-293 cell lysate
    Lane 4 : A431 cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 64 kDa
    Observed band size: 65 kDa why is the actual band size different from the predicted?



    Lanes 1-4: Merged signal (red and green). Green - ab32115 observed at 65 kDa. Red - loading control ab8245 observed at 37 kDa.

     ab32115 Anti-Paxillin antibody [E228] was shown to specifically react with Paxillin in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264767 (knockout cell lysate ab257044) was used. Wild-type and Paxillin knockout samples were subjected to SDS-PAGE. ab32115 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) were incubated overnight at 4°C at 1 in 10000 dilution and 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-Paxillin antibody [E228] (ab32115)
    Western blot - Anti-Paxillin antibody [E228] (ab32115)
    All lanes : Anti-Paxillin antibody [E228] (ab32115) at 1/10000 dilution

    Lane 1 : Wild-type A431 whole cell lysate
    Lane 2 : PXN knockout A431 whole cell lysate
    Lane 3 : U-87 MG whole cell lysate
    Lane 4 : PC-3 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 64 kDa
    Observed band size: 65 kDa why is the actual band size different from the predicted?



    Lanes 1 - 4: Merged signal (red and green). Green - ab32115 observed at 65 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab32115 was shown to specifically react with PXN in wild-type A431 cells as signal was lost in PXN knockout cells. Wild-type and PXN knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab32115 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/10000 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.

     

  • Western blot - Anti-Paxillin antibody [E228] (ab32115)
    Western blot - Anti-Paxillin antibody [E228] (ab32115)
    All lanes : Anti-Paxillin antibody [E228] (ab32115) at 1/1000 dilution (purified)

    Lane 1 : HEK-293 (Human embryonic kidney) whole cell lysates
    Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
    Lane 3 : C2C12 (Mouse myoblasts myoblast) whole cell lysates
    Lane 4 : Rat-1 (Rat embryonic fibroblast) whole cell lysates

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

    Predicted band size: 64 kDa
    Observed band size: 60,64 kDa why is the actual band size different from the predicted?



    Based on the immunogen sequence blast, this antibody recognizes alpha, beta and gamma isoforms. The molecular weight observed is consistent with what has been described in the literature PMID: 9712867 and 20388733

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Paxillin antibody [E228] (ab32115)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Paxillin antibody [E228] (ab32115)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast carcinoma tissue sections labeling Paxillin with Purified ab32115 at 1:50 dilution (2.34 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
  • Immunocytochemistry/ Immunofluorescence - Anti-Paxillin antibody [E228] (ab32115)
    Immunocytochemistry/ Immunofluorescence - Anti-Paxillin antibody [E228] (ab32115)

    Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Paxillin with Purified ab32115 at 1:100 dilution (1.2 µg/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). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

  • Immunoprecipitation - Anti-Paxillin antibody [E228] (ab32115)
    Immunoprecipitation - Anti-Paxillin antibody [E228] (ab32115)

    ab32115 (purified) at 1:20 dilution (0.5µg) immunoprecipitating Paxillin in HEK-293 whole cell lysate.
    Lane 1 (input): HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate 10µg
    Lane 2 (+): ab32115 & HEK-293 whole cell lysate
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32115 in HEK-293 whole cell lysate
    For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
    Blocking and diluting buffer: 5% NFDM/TBST.

  • Flow Cytometry (Intracellular) - Anti-Paxillin antibody [E228] (ab32115)
    Flow Cytometry (Intracellular) - Anti-Paxillin antibody [E228] (ab32115)

    Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Paxillin with Purified ab32115 at 1/20 dilution (10µg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluorr® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). 

     

     

  • Immunocytochemistry/ Immunofluorescence - Anti-Paxillin antibody [E228] (ab32115)
    Immunocytochemistry/ Immunofluorescence - Anti-Paxillin antibody [E228] (ab32115)

    Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Paxillin with ab32115 at 1/100 (1 μg/ml). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000, 2 μg/ml) was used as the secondary antibody. The cells were counterstained with ab195889, anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1/200, 2.5 μg/ml. Nuclei counterstained with DAPI (blue). 

    Confocal image showing membranous staining on HeLa cells.

  • Western blot - Anti-Paxillin antibody [E228] (ab32115)
    Western blot - Anti-Paxillin antibody [E228] (ab32115)
    All lanes : Anti-Paxillin antibody [E228] (ab32115) at 1/1000 dilution

    Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
    Lane 2 : HEK-293 (human embryonic kidney) whole cell lysates

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 64 kDa
    Observed band size: 64 kDa


    Exposure time: 3 minutes


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

  • Immunocytochemistry/ Immunofluorescence - Anti-Paxillin antibody [E228] (ab32115)
    Immunocytochemistry/ Immunofluorescence - Anti-Paxillin antibody [E228] (ab32115)Image from Beheshti Zavareh R et al., PLoS One. 2012;7(9):e43721. doi: 10.1371/journal.pone.0043721. Epub 2012 Sep 5. Fig 3.; doi:10.1371/journal.pone.0043721; September 5, 2012, PLoS ONE 7(9): e43721.

    Immunofluorescence analysis of HeLa cells, staining Paxillin with ab32115.

    Cells on the right were treated with MGAT1 shRNA. Cells were fixed with 2% paraformaldehyde, permeabilized using 0.2% Triton-X-100 and blocked by 5% BSA for 1 hour. Cells were incubated with primary antibody (1/400) overnight at 4°C. A FITC-conjugated donkey anti-rabbit IgG (1/500) was used as the secondary antibody.

  • Western blot - Anti-Paxillin antibody [E228] (ab32115)
    Western blot - Anti-Paxillin antibody [E228] (ab32115)
    All lanes : Anti-Paxillin antibody [E228] (ab32115) at 1/10000 dilution

    Lane 1 : untreated NIH 3T3 cell lysate
    Lane 2 : PDGF treated NIH 3T3 cell lysate

    Predicted band size: 64 kDa
    Observed band size: 64 kDa

  • ELISA - Anti-Paxillin antibody [E228] (ab32115)
    ELISA - Anti-Paxillin antibody [E228] (ab32115)

    Direct ELISA antigen dose-response curve using ab32115 at 0-1000 ng/mL. Antigen (human Paxillin phospho Y31 peptide/ unmodified peptide) concentration of 1000 ng/mL. An alkaline phosphatase-conjugated goat anti-rabbit IgG (H+L) (1/2500) was used as the secondary antibody.

  • Dot Blot - Anti-Paxillin antibody [E228] (ab32115)
    Dot Blot - Anti-Paxillin antibody [E228] (ab32115)

    Dot blot analysis of Paxillin (pY31) peptide (Lane 1) and Paxillin non-phospho peptide (Lane 2) labelling Paxillin with ab32115 at a dilution of 1/1000. ab97051 (Peroxidase conjugated goat anti-rabbit IgG (H+L)) was used as the secondary antibody at a dilution of 1/100000.

    Blocking and dilution buffer: 5% NFDM/TBST.

    Exposure time: 3 minutes.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Paxillin antibody [E228] (ab32115)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Paxillin antibody [E228] (ab32115)

    Immunohistochemical analysis of Paxillin expression in paraffin-embedded human colon carcinoma using 1/100 ab32115.

  • Anti-Paxillin antibody [E228] (ab32115)
    Anti-Paxillin antibody [E228] (ab32115)

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

References (8)

Publishing research using ab32115? Please let us know so that we can cite the reference in this datasheet.

ab32115 has been referenced in 8 publications.

  • Zhang M  et al. Sea cucumber Cucumaria frondosa fucoidan inhibits osteosarcoma adhesion and migration by regulating cytoskeleton remodeling. Oncol Rep 44:469-476 (2020). PubMed: 32467988
  • Gong X  et al. Expression of ILK in renal stroma is essential for multiple aspects of renal development. Am J Physiol Renal Physiol 315:F374-F385 (2018). PubMed: 29638158
  • Tsai CS  et al. Thrombomodulin regulates monocye differentiation via PKCd and ERK1/2 pathway in vitro and in atherosclerotic artery. Sci Rep 6:38421 (2016). WB ; Human . PubMed: 27910925
  • Sausgruber N  et al. Tyrosine phosphatase SHP2 increases cell motility in triple-negative breast cancer through the activation of SRC-family kinases. Oncogene N/A:N/A (2014). WB . PubMed: 24931162
  • Beheshti Zavareh R  et al. Suppression of cancer progression by MGAT1 shRNA knockdown. PLoS One 7:e43721 (2012). ICC/IF ; Human . PubMed: 22957033
  • Lin MY  et al. Biophysical stimulation induces demyelination via an integrin-dependent mechanism. Ann Neurol 72:112-23 (2012). PubMed: 22829273
  • Caltagarone J  et al. Paxillin and hydrogen peroxide-inducible clone 5 expression and distribution in control and Alzheimer disease hippocampi. J Neuropathol Exp Neurol 69:356-71 (2010). IHC-P ; Human . PubMed: 20448481
  • Wang H  et al. PRL-3 down-regulates PTEN expression and signals through PI3K to promote epithelial-mesenchymal transition. Cancer Res 67:2922-6 (2007). WB . PubMed: 17409395

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