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  1. Link

    syk-antibody-syk-01-ab3993.pdf

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Cardiovascular Blood Platelets
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Validated using a knockout cell line

Anti-Syk antibody [SYK-01] (ab3993)

  • Datasheet
  • SDS
Reviews (2)Q&A (2)References (22)

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Western blot - Anti-Syk antibody [SYK-01] (ab3993)
  • Western blot - Anti-Syk antibody [SYK-01] (ab3993)
  • Flow Cytometry - Anti-Syk antibody [SYK-01] (ab3993)

Key features and details

  • Mouse monoclonal [SYK-01] to Syk
  • Suitable for: WB, Flow Cyt
  • Knockout validated
  • Reacts with: Rat, Human
  • Isotype: IgG1

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Overview

  • Product name

    Anti-Syk antibody [SYK-01]
    See all Syk primary antibodies
  • Description

    Mouse monoclonal [SYK-01] to Syk
  • Host species

    Mouse
  • Specificity

    The antibody reacts with Protein tyrosine kinase p72Syk (Syk family tyrosine-specificphospho-transferase).
  • Tested applications

    Suitable for: WB, Flow Cytmore details
    Unsuitable for: ICC
  • Species reactivity

    Reacts with: Rat, Human
  • Immunogen

    Recombinant fragment, corresponding to amino acids 5-360 of Human Syk.

  • Positive control

    • Flow Cyt: Ramos cells WB: Ramos cell lysate, Rat basophilic leukemia cell lysate, HeLa cell lysate; HEK-293T cell lysate, HAP1 cell lysate.
  • General notes

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

    If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

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 or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.40
    Preservative: 0.097% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    SYK-01
  • Isotype

    IgG1
  • Research areas

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

Associated products

  • Compatible Secondaries

    • Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)
    • Goat Anti-Mouse IgG H&L (HRP) (ab205719)
    • Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879)
  • Conjugation kits

    • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
    • APC Conjugation Kit - Lightning-Link® (ab201807)
  • Isotype control

    • Mouse IgG1, kappa monoclonal [15-6E10A7] - Isotype Control (ab170190)
    • Mouse IgG1, Kappa Monoclonal [B11/6] - Isotype Control (ab91353)
  • Recombinant Protein

    • Recombinant human Syk protein (ab60886)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab3993 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)
Use a concentration of 1 - 2 µg/ml.
Flow Cyt
Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

Notes
WB
Use a concentration of 1 - 2 µg/ml.
Flow Cyt
Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

Application notes
Is unsuitable for ICC.

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: 25155 Rat
    • Omim: 600085 Human
    • SwissProt: P43405 Human
    • SwissProt: Q64725 Rat
    • Unigene: 371720 Human
    • Unigene: 87407 Rat
    • 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 [SYK-01] (ab3993)
      Western blot - Anti-Syk antibody [SYK-01] (ab3993)
      All lanes : Anti-Syk antibody [SYK-01] (ab3993) at 1 µg/ml

      Lane 1 : Wild-type HEK-293T cell lysate
      Lane 2 : SYK knockout HEK-293T cell lysate
      Lane 3 : HAP1 cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Observed band size: 72,73 kDa why is the actual band size different from the predicted?



      False colour image of Western blot: Anti-Syk antibody [SYK-01] staining at 1 ug/ml, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab3993 was shown to bind specifically to Syk. A band was observed at 72/73 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-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

    • Western blot - Anti-Syk antibody [SYK-01] (ab3993)
      Western blot - Anti-Syk antibody [SYK-01] (ab3993)
      Lanes 1-2 : Anti-Syk antibody [SYK-01] (ab3993) at 2 µg/ml
      Lanes 3-4 : anti-Human Cytokeratin 18


      Lane 1 : Ramos (human Burkitt's lymphoma cell line) cell lysate
      Lane 2 : RBL (rat basophilic leukemia cell line) cell lysate
      Lanes 3-4 : HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate
      Lane 5 : negative control

      Performed under non-reducing conditions.
    • Flow Cytometry - Anti-Syk antibody [SYK-01] (ab3993)
      Flow Cytometry - Anti-Syk antibody [SYK-01] (ab3993)

      Overlay histogram showing Ramos (Human Burkitt's lymphoma cell line) cells stained with ab3993 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab3993, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Ramos cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

    Protocols

    • Western blot protocols
    • Flow cytometry protocols
    • Immunocytochemistry & immunofluorescence protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (22)

    Publishing research using ab3993? Please let us know so that we can cite the reference in this datasheet.

    ab3993 has been referenced in 22 publications.

    • Yu Y  et al. Schistosome eggs stimulate reactive oxygen species production to enhance M2 macrophage differentiation and promote hepatic pathology in schistosomiasis. PLoS Negl Trop Dis 15:e0009696 (2021). PubMed: 34398890
    • Lamb DJ  et al. Inhibition of SYK kinase does not confer a pro-proliferative or pro-invasive phenotype in breast epithelium or breast cancer cells. Oncotarget 11:1257-1272 (2020). PubMed: 32292575
    • Li C  et al. The Mincle/Syk/NF-?B Signaling Circuit Is Essential for Maintaining the Protumoral Activities of Tumor-Associated Macrophages. Cancer Immunol Res 8:1004-1017 (2020). PubMed: 32532809
    • Bergsma A  et al. Regulation of cytoskeleton and adhesion signaling in osteoclasts by tetraspanin CD82. Bone Rep 10:100196 (2019). PubMed: 30788390
    • Gladkikh AA  et al. Comparison of the mRNA expression profile of B-cell receptor components in normal CD5-high B-lymphocytes and chronic lymphocytic leukemia: a key role of ZAP70. Cancer Med 6:2984-2997 (2017). PubMed: 29125235
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-4 of 4 Abreviews or Q&A

    Immunohistochemistry (Frozen sections) abreview for Anti-Syk antibody [SYK-01]

    Inconclusive
    Abreviews
    Abreviews
    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Mouse Tissue sections (Brain)
    Permeabilization
    Yes - 0.1% Tx
    Specification
    Brain
    Blocking step
    BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 24°C
    Fixative
    Paraformaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Dec 04 2018

    Western blot abreview for Anti-Syk antibody [SYK-01]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HUVEC, RAEC, RLMVEC)
    Loading amount
    10 µg
    Specification
    HUVEC, RAEC, RLMVEC
    Blocking step
    Other as blocking agent for 30 minute(s) · Concentration: 100%
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Aug 25 2006

    Question

    Does this antibody cross-react with Zap70?

    Read More

    Abcam community

    Verified customer

    Asked on Apr 19 2006

    Answer

    Thank you for your enquiry. This antibody has not been tested to cross react with Zap70. I hope this information helps. Please do not hesitate to contact us if you need anything further.

    Read More

    Abcam Scientific Support

    Answered on Apr 25 2006

    Question

    BATCH NUMBER 99152 DESCRIPTION OF THE PROBLEM Target protein is not precipitated SAMPLE mouse primary B cell lysate PRIMARY ANTIBODY antibody for developing WB: anti-Syk N19 from Santa Cruz rabbit 1:1000 1h RT (this antibody has worked very well for WB before and it also recognized the protein in the whole cell lysate control which was loaded in paralell, so I am sure, the lysate and the detection antibody are fine) SECONDARY ANTIBODY rabbit-HRP, Amersham, 1:1000 1h RT DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED whole cell lysate control (aliquot removed from lysate before IP) negative control with mouse IgG + protein G sepharose (no signal) positive control with Syk N19 agarose conjugate from Santa Cruz (good signal) ANTIBODY STORAGE CONDITIONS aliquots at -80 SAMPLE PREPARATION buffer for cell lysis, IP and IP wash: 20mM Hepes pH7.5 150mM NaCl 5mM EDTA 1% NP40 + protease and phosphatase inhibitors after last wash, boiled in SDS-sample buffer and loaded on SDS-PAGE MATRIX USED Protein G Sepharose DETERGENT 1% NP40 AMOUNT OF PROTEIN LOADED 100ug for IP ELECTROPHORESIS/GEL CONDITIONS 8% gel SDS-PAGE HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes ADDITIONAL NOTES I don't see any point in repeating the experiment as it is, because my controls all worked well. I am also sure that the protein G Sepharose worked, because I can see the antibody heavy chain in a Ponceau stain of the membrane. I specifically need an antibody for Syk IP which is of a species other than rabbit and it has to recognize the mouse Syk protein. From your datasheet, I expected that your product ab3993 should match these criteria. I normally have very good experiance with your products, so I am surprised that it does not work (it worked fine for WB, but I specifically want it for IP). I would be glad for any helpful advice or for an alternative product.

    Read More

    Abcam community

    Verified customer

    Asked on Mar 21 2005

    Answer

    Thank you for your enquiry and for taking your time to fill in the on-line questionnaire. We are very sorry to hear that you are having difficulty with this antibody. We would like to advise you to take a look at the following publication: http://www.jem.org/cgi/content/full/191/1/47 The following information can be found in this paper: Isolation of GEMs, Immunoprecipitations, Western Blots, and In-Gel Kinase Assays. GEMs were isolated from transfected 125I-IgE–labeled RBL-2H3 cells as described previously, except that 0.1% Triton X-100 was used for cell solubilization. Fractions were collected, and aliquots were counted for 125I-IgE distribution, then diluted 1:1 with lysis buffer and subjected sequentially to Vav, LAT, or Fc RI immunoprecipitation or to precipitation of all proteins with 20% trichloroacetic acid (TCA). For immunoprecipitations, lysis buffer (which for solubilization of GEMs included 0.5% ß-octyl glucoside) was added for 30 min at 4°C. Lysates were incubated with protein A or protein G Sepharose–bound antibodies to SLP-76, Vav, JNK1, Syk, Grb2, or LAT overnight at 4°C as indicated. SDS-solubilized proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with 4G10 antibody. For TCA precipitates, recovered pellets were washed once in ice-cold acetone, dried, and resuspended in 50 µl of 1% SDS in 50 mM Tricine and 50 µl of twofold-concentrated SDS sample buffer. Proteins were resolved and probed with antibodies to Grb2, LAT, Lyn, paxillin, Rac1, SLP-76, Syk, and Vav as indicated. This antibody has been tested for IP application by one of our collaborators. We would like to confirm that this product works well in IP but in our collaborator’s routine experiments, he used rabbit polyclonal which has higher capacity to immunoprecipitate. As we understand from your e-mail, SCBT Syk-agarose beads works in IP in comparison to the protocol he used with our Syk. We can confirm that immunosorbents like this (Syk-agarose) are more efficient then "classical" protein A/G mediated IP. We hope this information is useful for you. Should you still need further assistance or help, then please do not hesitate to contact us again.

    Read More

    Abcam Scientific Support

    Answered on Mar 31 2005

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
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