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

Reviews (1)Q&A (2)References (16)

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: ICC/IF, WB, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

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

  • Positive control
    • EB1- cells

Properties

Applications

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
ICC/IF Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml.
Flow Cyt Use 1µg for 106 cells.

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

 

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.
  • Information by UniProt
  • Database links
    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 [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.
  • Immunocytochemistry/ Immunofluorescence - Anti-Syk antibody [SYK-01] (ab3993)
    Immunocytochemistry/ Immunofluorescence - Anti-Syk antibody [SYK-01] (ab3993)

    Immunofluorescence staining of Syk in human HeLa (human epithelial cell line from cervix adenocarcinoma) cell line using ab3993 (green) at 5 µg/ml. Actin cytoskeleton was decorated by phalloidin (red) and cell nuclei stained with DAPI (blue).

  • Immunocytochemistry/ Immunofluorescence - Anti-Syk antibody [SYK-01] (ab3993)
    Immunocytochemistry/ Immunofluorescence - Anti-Syk antibody [SYK-01] (ab3993)

    Immunofluorescence staining of Syk in human primary fibroblasts using ab3993 (green) at 5 μg/ml. Actin cytoskeleton was decorated by phalloidin (red) and cell nuclei stained with DAPI (blue).

  • Western blot - Anti-Syk antibody [SYK-01] (ab3993)
    Western blot - Anti-Syk antibody [SYK-01] (ab3993)Image from Zou W et al, J Biol Chem. 2009 Jul 10;284(28):18833-9. Epub 2009 May 6, Fig 1. DOI 10.1074/jbc.M109.012385
    Transduced Syk-/- BMMs, cultured in 50 ng/ml M-CSF for 3 days, were serum- and cytokine-starved overnight. The cells were then exposed to either 100 ng/ml M-CSF with time. Signaling molecules were identified by immunoblotting. Actin serves as a loading control.
    Cultured cells were washed twice with ice-cold PBS and lysed in RIPA buffer containing 20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm ß-glycerophosphate, 1 mm Na3VO4, 1 mm NaF, and 1× protease inhibitor mixture. After incubation on ice for 10 minutes, cell lysates were clarified by centrifugation at 15,000 rpm for 10 minutes. 40µg of total lysates were subjected to 8% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Filters were blocked in 0.1% casein in PBS for 1 hour and incubated with primary antibodies at 4 °C overnight followed by probing with fluorescence-labeled secondary antibodies. Proteins were detected with the Odyssey infr
  • Flow Cytometry - Anti-Syk antibody [SYK-01] (ab3993)
    Flow Cytometry - Anti-Syk antibody [SYK-01] (ab3993)
    Overlay histogram showing Ramos 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.

References

This product has been referenced in:
  • Ho CM  et al. Inhibition of IKKa by BAY61-3606 Reveals IKKa-Dependent Histone H3 Phosphorylation in Human Cytomegalovirus Infected Cells. PLoS One 11:e0150339 (2016). WB . Read more (PubMed: 26930276) »
  • Shin HS  et al. Crosstalk among IL-23 and DNAX activating protein of 12 kDa-dependent pathways promotes osteoclastogenesis. J Immunol 194:316-24 (2015). Read more (PubMed: 25452564) »
See all 16 Publications for this product

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

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

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Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
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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%
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Abcam user community

Verified customer

Submitted Aug 25 2006

Question

Does this antibody cross-react with Zap70?

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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.

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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.

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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.

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