Recombinant 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
Related conjugates and formulations
Overview
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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 -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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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.
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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
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
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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...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
E228 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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KO cell lines
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KO cell lysates
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Positive Controls
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 |
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WB |
1/10000. Predicted molecular weight: 64 kDa.
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ICC/IF |
1/100.
For unpurified use at 1/250 |
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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 |
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IP |
1/20.
For unpurified use at 1/100 |
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Flow Cyt (Intra) |
1/20.
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Dot blot |
1/1000.
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ELISA |
Use at an assay dependent concentration.
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Notes |
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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
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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
modificationsPhosphorylated 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. - Information by UniProt
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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
see all -
Alternative names
- FLJ16691 antibody
- FLJ23042 antibody
- Paired box protein Pax 1 antibody
see all
Images
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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.
Performed under reducing conditions.
Predicted band size: 64 kDa
Observed band size: 65 kDa why is the actual band size different from the predicted?
Exposure time: 10 secondsLanes 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.
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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
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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.
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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.
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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. -
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).
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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.
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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 minutesBlocking and diluting buffer and concentration: 5% NFDM/TBST.
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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. -
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 -
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.
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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.
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Immunohistochemical analysis of Paxillin expression in paraffin-embedded human colon carcinoma using 1/100 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.
Datasheets and documents
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SDS download
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Datasheet download
References (12)
ab32115 has been referenced in 12 publications.
- Cheng P et al. Nidogen1-enriched extracellular vesicles accelerate angiogenesis and bone regeneration by targeting Myosin-10 to regulate endothelial cell adhesion. Bioact Mater 12:185-197 (2022). PubMed: 35310379
- Hauke M et al. Active RhoA Exerts an Inhibitory Effect on the Homeostasis and Angiogenic Capacity of Human Endothelial Cells. J Am Heart Assoc 11:e025119 (2022). PubMed: 35699166
- Huang Z et al. Identification of Paxillin as a Prognostic Factor for Glioblastoma via Integrated Bioinformatics Analysis. Biomed Res Int 2022:7171126 (2022). PubMed: 35782068
- Finney AC et al. EphA2 signaling within integrin adhesions regulates fibrillar adhesion elongation and fibronectin deposition. Matrix Biol 103-104:1-21 (2021). PubMed: 34537369
- 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