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    syk-antibody-ep573y-ab40781.pdf

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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Syk antibody [EP573Y] (ab40781)

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Western blot - Anti-Syk antibody [EP573Y] (ab40781)
  • Western blot - Anti-Syk antibody [EP573Y] (ab40781)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Syk antibody [EP573Y] (ab40781)
  • Immunocytochemistry/ Immunofluorescence - Anti-Syk antibody [EP573Y] (ab40781)
  • Flow Cytometry (Intracellular) - Anti-Syk antibody [EP573Y] (ab40781)
  • Western blot - Anti-Syk antibody [EP573Y] (ab40781)
  • Western blot - Anti-Syk antibody [EP573Y] (ab40781)
  • Immunocytochemistry/ Immunofluorescence - Anti-Syk antibody [EP573Y] (ab40781)
  • Western blot - Anti-Syk antibody [EP573Y] (ab40781)
  • Western blot - Anti-Syk antibody [EP573Y] (ab40781)
  • Flow Cytometry (Intracellular) - Anti-Syk antibody [EP573Y] (ab40781)
  • Anti-Syk antibody [EP573Y] (ab40781)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP573Y] to Syk
  • Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

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Overview

  • Product name

    Anti-Syk antibody [EP573Y]
    See all Syk primary antibodies
  • Description

    Rabbit monoclonal [EP573Y] to Syk
  • 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: Flow Cyt (Intra), WB, IHC-P, ICC/IFmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human Syk aa 300-400. The exact sequence is proprietary.

  • Positive control

    • WB: K562, Daudi, WEHI-231 and Raji cell lysates. Human, mouse and rat bone marrow lysates. IHC-P: Human spleen. ICC/IF: Raji and Daudi cells. Flow Cyt (intra): Raji and K562 cells.
  • 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. 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: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EP573Y
  • Isotype

    IgG
  • Research areas

    • Cardiovascular
    • Blood
    • Platelets
    • Signal Transduction
    • Protein Phosphorylation
    • Tyrosine Kinases
    • Other
    • Immunology
    • Innate Immunity
    • Mast Cells
    • Neuroscience
    • Processes

Associated products

  • Alternative Versions

    • Anti-Syk antibody [EP573Y] - BSA and Azide free (ab190176)
    • Alexa Fluor® 488 Anti-Syk antibody [EP573Y] (ab198938)
    • HRP Anti-Syk antibody [EP573Y] (ab198940)
  • Compatible Secondaries

    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • Recombinant Protein

    • Recombinant human Syk protein (ab60886)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab40781 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
Flow Cyt (Intra)
1/70.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

WB
1/1000 - 1/5000. Detects a band of approximately 72 kDa (predicted molecular weight: 72 kDa).
IHC-P
1/5000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

For unpurified use at 1/100 - 1/250.

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

ICC/IF
1/100 - 1/250.
Notes
Flow Cyt (Intra)
1/70.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

WB
1/1000 - 1/5000. Detects a band of approximately 72 kDa (predicted molecular weight: 72 kDa).
IHC-P
1/5000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

For unpurified use at 1/100 - 1/250.

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

ICC/IF
1/100 - 1/250.

Target

  • Function

    Non-receptor tyrosine kinase which mediates signal transduction downstream of a variety of transmembrane receptors including classical immunoreceptors like the B-cell receptor (BCR). Regulates several biological processes including innate and adaptive immunity, cell adhesion, osteoclast maturation, platelet activation and vascular development. Assembles into signaling complexes with activated receptors at the plasma membrane via interaction between its SH2 domains and the receptor tyrosine-phosphorylated ITAM domains. The association with the receptor can also be indirect and mediated by adapter proteins containing ITAM or partial hemITAM domains. The phosphorylation of the ITAM domains is generally mediated by SRC subfamily kinases upon engagement of the receptor. More rarely signal transduction via SYK could be ITAM-independent. Direct downstream effectors phosphorylated by SYK include VAV1, PLCG1, PI-3-kinase, LCP2 and BLNK. Initially identified as essential in B-cell receptor (BCR) signaling, it is necessary for the maturation of B-cells most probably at the pro-B to pre-B transition. Activated upon BCR engagement, it phosphorylates and activates BLNK an adapter linking the activated BCR to downstream signaling adapters and effectors. It also phosphorylates and activates PLCG1 and the PKC signaling pathway. It also phosphorylates BTK and regulates its activity in B-cell antigen receptor (BCR)-coupled signaling. In addition to its function downstream of BCR plays also a role in T-cell receptor signaling. Plays also a crucial role in the innate immune response to fungal, bacterial and viral pathogens. It is for instance activated by the membrane lectin CLEC7A. Upon stimulation by fungal proteins, CLEC7A together with SYK activates immune cells inducing the production of ROS. Also activates the inflammasome and NF-kappa-B-mediated transcription of chemokines and cytokines in presence of pathogens. Regulates neutrophil degranulation and phagocytosis through activation of the MAPK signaling cascade. Also mediates the activation of dendritic cells by cell necrosis stimuli. Also involved in mast cells activation. Also functions downstream of receptors mediating cell adhesion. Relays for instance, integrin-mediated neutrophils and macrophages activation and P-selectin receptor/SELPG-mediated recruitment of leukocytes to inflammatory loci. Plays also a role in non-immune processes. It is for instance involved in vascular development where it may regulate blood and lymphatic vascular separation. It is also required for osteoclast development and function. Functions in the activation of platelets by collagen, mediating PLCG2 phosphorylation and activation. May be coupled to the collagen receptor by the ITAM domain-containing FCER1G. Also activated by the membrane lectin CLEC1B that is required for activation of platelets by PDPN/podoplanin. Involved in platelet adhesion being activated by ITGB3 engaged by fibrinogen.
  • Tissue specificity

    Widely expressed in hematopoietic cells (at protein level). Within the B-cells compartment it is for instance expressed for pro-B-cells to plasma cells.
  • Sequence similarities

    Belongs to the protein kinase superfamily. Tyr protein kinase family. SYK/ZAP-70 subfamily.
    Contains 1 protein kinase domain.
    Contains 2 SH2 domains.
  • Domain

    The SH2 domains mediate the interaction of SYK with the phosphorylated ITAM domains of transmembrane proteins. Some proteins like CLEC1B have a partial ITAM domain (also called hemITAM) containing a single YxxL motif. The interaction with SYK requires CLEC1B homodimerization.
  • Post-translational
    modifications

    Ubiquitinated by CBLB after BCR activation; which promotes proteasomal degradation.
    Autophosphorylated. Phosphorylated on tyrosine residues by LYN following receptors engagement. Phosphorylation on Tyr-323 creates a binding site for CBL, an adapter protein that serves as a negative regulator of BCR-stimulated calcium ion signaling. Phosphorylation at Tyr-348 creates a binding site for VAV1. Phosphorylation on Tyr-348 and Tyr-352 enhances the phosphorylation and activation of phospholipase C-gamma and the early phase of calcium ion mobilization via a phosphoinositide 3-kinase-independent pathway (By similarity). Phosphorylation on Ser-297 is very common, it peaks 5 minutes after BCR stimulation, and creates a binding site for YWHAG. Phosphorylation at Tyr-630 creates a binding site for BLNK. Dephosphorylated by PTPN6.
  • Cellular localization

    Cell membrane. Cytoplasm, cytosol.
  • Target information above from: UniProt accession P43405 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 6850 Human
    • Entrez Gene: 20963 Mouse
    • Entrez Gene: 25155 Rat
    • Omim: 600085 Human
    • SwissProt: P43405 Human
    • SwissProt: P48025 Mouse
    • SwissProt: Q64725 Rat
    • Unigene: 371720 Human
    • Unigene: 375031 Mouse
    • Unigene: 87407 Rat
    see all
  • Alternative names

    • EC 2.7.10.2 antibody
    • kinase Syk antibody
    • KSYK antibody
    • KSYK_HUMAN antibody
    • p72-Syk antibody
    • p72syk antibody
    • Spleen tyrosine kinase antibody
    • Syk antibody
    • Tyrosine protein kinase SYK antibody
    • Tyrosine-protein kinase SYK antibody
    see all

Images

  • Western blot - Anti-Syk antibody [EP573Y] (ab40781)
    Western blot - Anti-Syk antibody [EP573Y] (ab40781)
    All lanes : Anti-Syk antibody [EP573Y] (ab40781) at 1/1000 dilution

    Lane 1 : Wild-type HEK-293T cell lysate
    Lane 2 : SYK knockout HEK-293T cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 72 kDa
    Observed band size: 70 kDa why is the actual band size different from the predicted?



    False colour image of Western blot: Anti-Syk antibody [EP573Y] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab40781 was shown to bind specifically to Syk. A band was observed at 70/72 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in SYK knockout cell line ab282649 (knockout cell lysate ab283048). To generate this image, wild-type and SYK knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

  • Western blot - Anti-Syk antibody [EP573Y] (ab40781)
    Western blot - Anti-Syk antibody [EP573Y] (ab40781)

    Lane 1: Wild-type HAP1 cell lysate (40 µg)
    Lane 2: SYK knockout HAP1 cell lysate (40 µg)
    Lane 3: K562 cell lysate (40 µg)
    Lane 4: Raji cell lysate (40 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab40781 observed at 75 kDa. Red - loading control, ab8245, observed at 37 kDa.

    ab40781 was shown to specifically react with Syk when Syk knockout samples were used. Wild-type and Syk knockout samples were subjected to SDS-PAGE. Ab40781 and ab8245 (loading control to GAPDH) were diluted at 1/5000 and 1/10000 dilution respectively and incubated overnight at 4C. 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 hour at room temperature before imaging.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Syk antibody [EP573Y] (ab40781)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Syk antibody [EP573Y] (ab40781)

    Immunohistochemistry of paraffin embedded Human spleen tissue section labelling Syk with ab40781 at 1:5000 dilution (0.44 μg/ml).  Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). A ready to use ImmunoHistoProbe one step HRP Polymer (ready to use) was used as a secondary antibody at 1:0 dilution. PBS instead of primary antibody was used for negative control. Hematoxylin was used as a counterstain.

  • Immunocytochemistry/ Immunofluorescence - Anti-Syk antibody [EP573Y] (ab40781)
    Immunocytochemistry/ Immunofluorescence - Anti-Syk antibody [EP573Y] (ab40781)

    Immunocytochemistry/ Immunofluorescence analysis of Daudi (Human Burkitt's lymphoma lymphoblast) cells labeling Syk with Purified ab40781 at 1:150 (5 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 (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

  • Flow Cytometry (Intracellular) - Anti-Syk antibody [EP573Y] (ab40781)
    Flow Cytometry (Intracellular) - Anti-Syk antibody [EP573Y] (ab40781)

    Intracellular Flow Cytometry analysis of Raji (Human Burkitt's lymphoma B lymphocyte) cells labeling Syk with Purified ab40781 at 1/70 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 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). 

     

     

  • Western blot - Anti-Syk antibody [EP573Y] (ab40781)
    Western blot - Anti-Syk antibody [EP573Y] (ab40781)
    All lanes : Anti-Syk antibody [EP573Y] (ab40781) at 1/1000 dilution (Purified)

    Lane 1 : Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysates
    Lane 2 : Daudi (Human Burkitt's lymphoma lymphoblast) whole cell lysates
    Lane 3 : WEHI-231 (Mouse B cell lymphoma B lymphocyte ) 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: 72 kDa
    Observed band size: 72 kDa



    Blocking/Diluting buffer: 5% NFDM/TBST

  • Western blot - Anti-Syk antibody [EP573Y] (ab40781)
    Western blot - Anti-Syk antibody [EP573Y] (ab40781)
    Anti-Syk antibody [EP573Y] (ab40781) at 1/1000 dilution (Purified) + Human bone marrow lysates at 15 µg

    Secondary
    Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

    Predicted band size: 72 kDa
    Observed band size: 72 kDa



    Blocking/Diluting buffer: 5% NFDM/TBST

  • Immunocytochemistry/ Immunofluorescence - Anti-Syk antibody [EP573Y] (ab40781)
    Immunocytochemistry/ Immunofluorescence - Anti-Syk antibody [EP573Y] (ab40781)

    Immunocytochemistry/Immunofluorescence analysis of Raji (human Burkitt's lymphoma B lymphocyte) labelling Syk with ab40781 at a dilution of 1:100 dilution (7µg/ml). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat anti-rabbit IgG (Alexa Fluor® 488, ab150077) (1:1000 dilution (2 µg/ml)) was used as the secondary antibody. The cells were co-stained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5µg/ml). Nuclei counterstained with DAPI (blue). Control: PBS instead of the primary antibody.

  • Western blot - Anti-Syk antibody [EP573Y] (ab40781)
    Western blot - Anti-Syk antibody [EP573Y] (ab40781)
    Anti-Syk antibody [EP573Y] (ab40781) at 1/2000 dilution + Rat bone marrow lysate at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 72 kDa


    Exposure time: 50 seconds


    Blocking and diluting buffer: 5% NFDM/TBST. 

  • Western blot - Anti-Syk antibody [EP573Y] (ab40781)
    Western blot - Anti-Syk antibody [EP573Y] (ab40781)
    Anti-Syk antibody [EP573Y] (ab40781) at 1/5000 dilution + Mouse bone marrow lysate at 20 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Predicted band size: 72 kDa


    Exposure time: 3 minutes


    Blocking and diluting buffer: 5% NFDM/TBST.

  • Flow Cytometry (Intracellular) - Anti-Syk antibody [EP573Y] (ab40781)
    Flow Cytometry (Intracellular) - Anti-Syk antibody [EP573Y] (ab40781)

    Intracellular Flow Cytometry analysis of K562 (human chronic myelogenous leukemia)cells labeling Syk with unpurified ab40781 at 1/70 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) (1/2000 dilution) was used as the secondary antibody. Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control. 

     

     

  • Anti-Syk antibody [EP573Y] (ab40781)
    Anti-Syk antibody [EP573Y] (ab40781)

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

  • Datasheet download

    Download

References (17)

Publishing research using ab40781? Please let us know so that we can cite the reference in this datasheet.

ab40781 has been referenced in 17 publications.

  • Gong W  et al. CCL4-mediated targeting of spleen tyrosine kinase (Syk) inhibitor using nanoparticles alleviates inflammatory bowel disease. Clin Transl Med 11:e339 (2021). PubMed: 33634985
  • Sun J  et al. Anti-allergic actions of a Chinese patent medicine, huoxiangzhengqi oral liquid, in RBL-2H3 cells and in mice. Pharm Biol 59:672-682 (2021). PubMed: 34078224
  • Hou YB  et al. The antipsychotic drug pimozide inhibits IgE-mediated mast cell degranulation and migration. Int Immunopharmacol 84:106500 (2020). PubMed: 32311669
  • Zhao X  et al. Activation of C-Type Lectin Receptor and (RIG)-I-Like Receptors Contributes to Proinflammatory Response in Middle East Respiratory Syndrome Coronavirus-Infected Macrophages. J Infect Dis 221:647-659 (2020). PubMed: 31562757
  • Gudjonsson JE  et al. Contribution of plasma cells and B cells to hidradenitis suppurativa pathogenesis. JCI Insight 5:N/A (2020). PubMed: 32853177
View all Publications for this product

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