Anti-SHP2 (phospho S576) antibody (ab17940)

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Overview

  • Product name
    Anti-SHP2 (phospho S576) antibody
    See all SHP2 primary antibodies
  • Description
    Rabbit polyclonal to SHP2 (phospho S576)
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WBmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse, Rat
  • Immunogen

    The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human SHP2 that contains serine 576.

  • Positive control
    • Hek293 cells treated with PMA.

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: 7.30
    Preservative: 0.05% Sodium azide
    Constituents: PBS, 50% Glycerol, 0.1% BSA

    PBS is Ca2+ and Mg2+ free
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Purification notes
    Purified from rabbit serum by sequential epitope-specific chromatography. The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated SHP2. The final product is generated by affinity chromatography using a SHP2-derived peptide that is phosphorylated at serine 576.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab17940 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 1/1000. Detects a band of approximately 68 kDa (predicted molecular weight: 68 kDa).

Target

  • Function
    Acts downstream of various receptor and cytoplasmic protein tyrosine kinases to participate in the signal transduction from the cell surface to the nucleus.
  • Tissue specificity
    Widely expressed, with highest levels in heart, brain, and skeletal muscle.
  • Involvement in disease
    Defects in PTPN11 are the cause of LEOPARD syndrome type 1 (LEOPARD1) [MIM:151100]. It is an autosomal dominant disorder allelic with Noonan syndrome. The acronym LEOPARD stands for lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonic stenosis, abnormalities of genitalia, retardation of growth, and deafness.
    Defects in PTPN11 are the cause of Noonan syndrome type 1 (NS1) [MIM:163950]. Noonan syndrome (NS) is a disorder characterized by dysmorphic facial features, short stature, hypertelorism, cardiac anomalies, deafness, motor delay, and a bleeding diathesis. Some patients with Noonan syndrome type 1 develop multiple giant cell lesions of the jaw or other bony or soft tissues, which are classified as pigmented villomoduolar synovitis (PVNS) when occurring in the jaw or joints. Note=Mutations in PTPN11 account for more than 50% of the cases. Rarely, NS is associated with juvenile myelomonocytic leukemia (JMML). NS1 inheritance is autosomal dominant.
    Defects in PTPN11 are a cause of juvenile myelomonocytic leukemia (JMML) [MIM:607785]. JMML is a pediatric myelodysplastic syndrome that constitutes approximately 30% of childhood cases of myelodysplastic syndrome (MDS) and 2% of leukemia. It is characterized by leukocytosis with tissue infiltration and in vitro hypersensitivity of myeloid progenitors to granulocyte-macrophage colony stimulating factor.
    Defects in PTPN11 are a cause of metachondromatosis (MC) [MIM:156250]. It is a skeletal disorder with radiologic fetarures of both multiple exostoses and Ollier disease, characterized by the presence of multiple enchondromas and osteochondroma-like lesions.
  • Sequence similarities
    Belongs to the protein-tyrosine phosphatase family. Non-receptor class 2 subfamily.
    Contains 2 SH2 domains.
    Contains 1 tyrosine-protein phosphatase domain.
  • Domain
    The SH2 domains repress phosphatase activity. Binding of these domains to phosphotyrosine-containing proteins relieves this auto-inhibition, possibly by inducing a conformational change in the enzyme.
  • Post-translational
    modifications
    Phosphorylated on Tyr-546 and Tyr-584 upon receptor protein tyrosine kinase activation; which creates a binding site for GRB2 and other SH2-containing proteins.
  • Cellular localization
    Cytoplasm.
  • Information by UniProt
  • Database links
    see all
  • Alternative names
    • BPTP3 antibody
    • CFC antibody
    • JMML antibody
    • METCDS antibody
    • MGC14433 antibody
    • NS1 antibody
    • OTTHUMP00000166107 antibody
    • OTTHUMP00000166108 antibody
    • Protein tyrosine phosphatase 2 antibody
    • Protein tyrosine phosphatase 2C antibody
    • Protein tyrosine phosphatase non receptor type 11 antibody
    • Protein-tyrosine phosphatase 1D antibody
    • Protein-tyrosine phosphatase 2C antibody
    • PTN11_HUMAN antibody
    • PTP-1D antibody
    • PTP-2C antibody
    • PTP1D antibody
    • PTP2C antibody
    • PTPN11 antibody
    • SAP2 antibody
    • SH-PTP2 antibody
    • SH-PTP3 antibody
    • SH2 domain containing protein tyrosine phosphatase 2 antibody
    • SHP 2 antibody
    • SHP-2 antibody
    • Shp2 antibody
    • SHPTP2 antibody
    • SHPTP3 antibody
    • Syp antibody
    • Tyrosine-protein phosphatase non-receptor type 11 antibody
    see all

Images

  • Western blot - Anti-SHP2 (phospho S576) antibody (ab17940)
    Western blot - Anti-SHP2 (phospho S576) antibody (ab17940)

    Peptide Competition and Phosphatase Treatment

    Lysates prepared from Hek293 treated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either treated with lambda (λ) phosphatase (1) or left untreated (2-7), blocked with a 3% BSA-TBST buffer for one hour at room temperature, and incubated with SHP2 [pS576] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the immunogen (3), a generic phosphoserine-containing peptide (4), the phosphopeptide corresponding to SHP2 [pS585] (5), the phosphopeptide corresponding to SHP2 [pS591] (6), or, the phosphopeptide immunogen (7). After washing, membranes were incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal™ method.

    The data show that only the peptide corresponding to SHP2 [pS576] bl

References

ab17940 has not yet been referenced specifically in any publications.

Publishing research using ab17940? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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Question

Looking through both protocols, it seems that this enquiry and the other (CCEID:743720 - ab13948) are from the same customer. Is that correct? Ye, same customer. - First of all, I would like to clarify that ab17940 is not SOCS4 but SHP2 (phospho S576) - you say in your e-mail Rabbit Polyclonal to SOCS4(abcam)). --> Our customer had confused. Your are all right. - I would highly recommend using RIPA buffer and not TCA for precipitation. This buffer contains a cocktail of different proteinase inhibitors which help prevent the degradation of the target protein. TCA may destroy the epitipe(s) and can cause some detection problem. Actually, we have not used TCA protein precipitates for testing and characterizing this product (and any of our other antibodies), therefore we have no experience with this type of extraction. ---> She changed buffer with RIPA, but the results were same. - You have written in your e-mail: Incubation for 2h at room temp. or overnight at 4? Question: Could you please specify if it was a 2hr or overnight incubation? It is not clear from your message. She had tried to incubation both of them, and she had same results. - Since the antibody recognizes the phosphorylated SH2 protein, it is important to use 5% BSA as blocking agent rather than milk.Milk may cause high background staining. I would also suggest that the customer should use positive control i.e. Hek293 cells treated with PMA. She had used 5% BSA but no differece with milk. - Does the sample express high levels of phosphorylated SHP2? She do not have reference, but she had complaint with background. - Please also advise your customer to load at least 20-30 ug protein per lane. She had loaded 30ug per lane, but obtained same results. - Is the secondary antibody works properly? Has the customer used it successfully with another primary antibody? It would be advisable to run a no primary – only secondary antibody – control to see if the high background signal is still there. She had a good results using other primary antibody.

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Answer

Dr Bagrij is away from the office for a few days and I will do my best to help you to prevent delays for you and the customer. I understand that the customer has experienced problems with ab17940 and ab13948. You also sent to me today a complaint for ab3695 with a protocol very similar to the other protocols, is this the same customer? If so this makes me think there is a problem with the storage of the antibodies, the samples, or something gone off in the buffers used in all experiments or the preservation condition of the lysates has not been adequate. We have not received any other complaints about those antibodies. Can you please clarify if the customer is seeing the correct band plus high background or just high background? As Dr Bagrij mentioned it is possible that the customer's samples do not contain the phosphorylated form of SHP2 protein or the SOCS4 protein or that total SHP2 is not even in the samples. I would recommend to run the positive control of Hek293 cells treated with PMA (which induces the phosphorylation) and to make sure the protease inhibitors in the lysis buffer are fresh and of high concentration. What buffer and protease inhibitors were used? I was able to find the lysis buffer used with the HEK293 treated cell lysate, as you can see Triton or NP40 is essential to lyse the cells properly: 10 mM Tris, pH 7.4 100 mM NaCl 1 mM EDTA 1 mM EGTA 1 mM NaF 20 mM Na4P2O7 2 mM Na3VO4 0.1% SDS 0.5% sodium deoxycholate 1% Triton-X 100 10% glycerol 1 mM PMSF (made from a 0.3 M stock in DMSO) or 1 mM AEBSF (water soluble version of PMSF) 60 µg/mL aprotinin 10 µg/mL leupeptin 1 µg/mL pepstatin (alternatively, protease inhibitor cocktail such as Sigma Cat. # P2714 may be used). For ab13948 I am trying to find out the recommended positive control to help the customer. I would appreciate if you can provide the images of the blots before Dr Bagrij's recommendations and after, to help me diagnose the problem better. It is still very difficult at this stage to see what the problem might be and this will help me a lot. I appreciate your help with this matter and look forward to hearing from you,

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