Overview

  • Product name
    Anti-SHC (phospho S36) antibody [6E10]
    See all SHC primary antibodies
  • Description
    Mouse monoclonal [6E10] to SHC (phospho S36)
  • Host species
    Mouse
  • Specificity
    This antibody recognizes the 66kDa form of SHC protein phosphorylated on Ser36. It does not cross react with the non-phosphorylated form of SHC or with unrelated phosphorylation sites.
  • Tested applications
    Suitable for: WB, ELISA, IHC-P, ICC/IF, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic phosphopeptide corresponding to amino acids surrounding the Ser36 phosphorylation site of human SHC.

  • Positive control
    • HeLa cells stimulated with EGF

Properties

Applications

Our Abpromise guarantee covers the use of ab54518 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 Use a concentration of 0.1 - 1 µg/ml. Predicted molecular weight: 63 kDa.
ELISA Use a concentration of 0.05 µg/ml.
IHC-P Use at an assay dependent concentration. PubMed: 24273694
ICC/IF Use at an assay dependent concentration. PubMed: 21799744
Flow Cyt Use 2µg for 106 cells.

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

 

Target

  • Function
    Signaling adapter that couples activated growth factor receptors to signaling pathways. Participates in a signaling cascade initiated by activated KIT and KITLG/SCF. Isoform p46Shc and isoform p52Shc, once phosphorylated, couple activated receptor tyrosine kinases to Ras via the recruitment of the GRB2/SOS complex and are implicated in the cytoplasmic propagation of mitogenic signals. Isoform p46Shc and isoform p52Shc may thus function as initiators of the Ras signaling cascade in various non-neuronal systems. Isoform p66Shc does not mediate Ras activation, but is involved in signal transduction pathways that regulate the cellular response to oxidative stress and life span. Isoform p66Shc acts as a downstream target of the tumor suppressor p53 and is indispensable for the ability of stress-activated p53 to induce elevation of intracellular oxidants, cytochrome c release and apoptosis. The expression of isoform p66Shc has been correlated with life span (By similarity). Participates in signaling downstream of the angiopoietin receptor TEK/TIE2, and plays a role in the regulation of endothelial cell migration and sprouting angiogenesis.
  • Tissue specificity
    Widely expressed. Expressed in neural stem cells but absent in mature neurons.
  • Sequence similarities
    Contains 1 PID domain.
    Contains 1 SH2 domain.
  • Domain
    In response to a variety of growth factors, isoform p46Shc and isoform p52Shc bind to phosphorylated Trk receptors through their phosphotyrosine binding (PID) and/or SH2 domains. The PID and SH2 domains bind to specific phosphorylated tyrosine residues in the Asn-Pro-Xaa-Tyr(P) motif of the Trk receptors. Isoform p46Shc and isoform p52Shc are in turn phosphorylated on three tyrosine residues within the extended proline-rich domain. These phosphotyrosines act as docking site for GRB2 and thereby are involved in Ras activation.
  • Post-translational
    modifications
    Phosphorylated by activated epidermal growth factor receptor. Phosphorylated in response to FLT4 and KIT signaling. Isoform p46Shc and isoform p52Shc are phosphorylated on tyrosine residues of the Pro-rich domain. Isoform p66Shc is phosphorylated on Ser-36 by PRKCB upon treatment with insulin, hydrogen peroxide or irradiation with ultraviolet light (By similarity). Tyrosine phosphorylated in response to FLT3 signaling (By similarity). Tyrosine phosphorylated by activated PTK2B/PYK2 (By similarity). Tyrosine phosphorylated by ligand-activated ALK. Tyrosine phosphorylated by ligand-activated PDGFRB. Tyrosine phosphorylated by TEK/TIE2. May be tyrosine phosphorylated by activated PTK2/FAK1; tyrosine phosphorylation was seen in an astrocytoma biopsy, where PTK2/FAK1 kinase activity is high, but not in normal brain tissue. Isoform p52Shc dephosphorylation by PTPN2 may regulate interaction with GRB2.
  • Cellular localization
    Cytoplasm; Mitochondrion matrix. Localized to the mitochondria matrix. Targeting of isoform p46Shc to mitochondria is mediated by its first 32 amino acids, which behave as a bona fide mitochondrial targeting sequence. Isoform p52Shc and isoform p66Shc, that contain the same sequence but more internally located, display a different subcellular localization and Mitochondrion. In case of oxidative conditions, phosphorylation at 'Ser-36' of isoform p66Shc, leads to mitochondrial accumulation.
  • Information by UniProt
  • Database links
  • Alternative names
    • FLJ26504 antibody
    • p66 antibody
    • p66SHC antibody
    • SH2 domain protein C1 antibody
    • SHC (Src homology 2 domain containing) transforming protein 1 antibody
    • SHC 1 antibody
    • SHC A antibody
    • SHC adaptor protein 1 antibody
    • Shc antibody
    • SHC transforming protein 1 antibody
    • SHC transforming protein antibody
    • SHC-transforming protein 1 antibody
    • SHC-transforming protein 3 antibody
    • SHC-transforming protein A antibody
    • SHC1 antibody
    • SHC1_HUMAN antibody
    • SHCA antibody
    • Src homology 2 domain-containing-transforming protein C1 antibody
    see all

Images

  • All lanes : Anti-SHC (phospho S36) antibody [6E10] (ab54518) at 1 µg/ml

    Lane 1 : HeLa cells stimulated with EGF for 0 mins.
    Lane 2 : HeLa cells stimulated with EGF for 1 min.
    Lane 3 : HeLa cells stimulated with EGF for 5 mins.
    Lane 4 : HeLa cells stimulated with EGF for 15 mins.
    Lane 5 : HeLa cells stimulated with EGF for 30 mins.
    Lane 6 : HeLa cells stimulated with EGF for 60 mins.

    Lysates/proteins at 15 µl per lane.

    Developed using the ECL technique.

    Predicted band size: 63 kDa
    Observed band size: 67 kDa
    why is the actual band size different from the predicted?

  • Overlay histogram showing MCF7 cells stained with ab54518 (red line). The cells were fixed with 80% methanol (5 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 (ab54518, 2µg/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.

References

This product has been referenced in:
  • Halon-Golabek M  et al. hmSOD1 gene mutation-induced disturbance in iron metabolism is mediated by impairment of Akt signalling pathway. J Cachexia Sarcopenia Muscle N/A:N/A (2018). WB . Read more (PubMed: 29380557) »
  • D'Agostino M  et al. Role of miR-200c in Myogenic Differentiation Impairment via p66Shc: Implication in Skeletal Muscle Regeneration of Dystrophic mdx Mice. Oxid Med Cell Longev 2018:4814696 (2018). WB ; Mouse . Read more (PubMed: 29636844) »
See all 14 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Question
Answer

Thanks for notifying us regarding the lack of signal with ab54518. One important point of emphasis is to make sure you are treating your samples. Endogenous SHC phosphorylation at S36 is extremely low and may not even be detectable in certain samples. The following articles demonstrate this quite nicely: http://www.ncbi.nlm.nih.gov/pubmed/?term=p66+SHC+S36 http://circres.ahajournals.org/content/104/5/660.full#F6 I hope that this is helpful!

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Answer

Thank you for providing that extra information. It allows me to more fully understand what may be contributing to the problems observed.

May I ask, in the blot presented, what bands were derived from the abcam antibody and what were obtained with the other antibody? The band below the smeared band at ˜ 70 kDa, what size does this equate to and is this a loading control? What is loaded in each well (are some induced and some not)?

May I also ask, how was the gel prepared and run? Were they made in house or purchased precast?

Also, how was the gel run? For what time/voltage? And were the buffers used cooled?

With this information I will hopefully be able to provide a few suggestions which should help to improve the results observed so far.I look forward to receiving your reply.

Many thanks for your cooperation.

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Question

LOT NUMBER GR70052-1 ORDER NUMBER 342 DESCRIPTION OF THE PROBLEM Non-specific band the detection band is not defined it as a smear rather unspecific. SAMPLE cell lysate of human fibroblasts PRIMARY ANTIBODY 1 µg/ml in PBS-tween 3%BSA DETECTION METHOD ECL and ECL plus POSITIVE AND NEGATIVE CONTROLS USED Fibroblasts are a positive control for p66-SHC: we have already performed a western blot with human fibroblasts blotted with a phospho-p66SHC antibody from another company and we obtained the relative band (attached there’s the blot) All the analysed fibroblasts were between the 4th and the 7th passages ANTIBODY STORAGE CONDITIONS The antibody was aliquoted and stored at -20 ° C SAMPLE PREPARATION Samples are lysate 30 minutes on ice in RIPA buffer + DTT +NaVO4 +Complete protease inhibitors cocktail (Roche: 1 tablet in 50 ml RIPA buffer) Proteins are suspended in loading buffer: TRIS-HCl pH6.8, SDS, glycerol, bromophenol blue, beta-mercaptoethanol. Samples are heated 5 minutes to 95°C and loaded on the gel AMOUNT OF PROTEIN LOADED Protein loaded on the gel: 90 µg ELECTROPHORESIS/GEL CONDITIONS Reducing gel % of the gel: 10% TRANSFER AND BLOCKING CONDITIONS The proteins were transferred onto nitrocellulose membrane, blocked in TBS-Tween 3% BSA for 1 hour, then were made two washes of 10 min in TBS-Tween and then incubation with the primary antibody (1 µg / ml ) in TBS-tween 3% BSA under stirring at +4 ° C over night. Then the membrane was washed twice for 10 min with TBS-Tween, then occurred incubation with secondary antibody (1:1000) for 1 hour at room temperature, then 3 washes of 10 min and finally signal detection by chemiluminescence SECONDARY ANTIBODY (anti-mouse Dako) 1:1000 HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? none

Read More
Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

In order to understand the problem more fully could I please ask a few further questions:

1. The other anti-SHC antibody used, where was this from, manufacturer, catalogue number? Was the same secondary antibody used for both antibodies?

2. If not, what secondary antibody was used with ab54518(cataloguenumber) and had this secondary antibody been used successfully before?

3. Was the phosphorylation state induced in the fibroblast samples by treatment with EGF or similar?

Also, could you please reattach the image of the results obtained as this did not reach me.

If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I look forward to receiving your reply.

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Answer

Thank you for contacting us.

I afraid we do not supply those antibodies in pure PBS. If you are planning toplace an order for more than 10 units of each reference, I can still ask the labs if there would be any possibility to have a production batch in pure PBS.

Otherwise, I would suggest to purify the antibodies using our antibody purification kits https://www.abcam.com/ab102783 (1 purification) or https://www.abcam.com/ab102784 (3 purifications).

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Question
Answer

DISCOUNT CODE: ***
Expiration date: June 29th, 2012

I am very pleased to hear you would like to accept our offer and test ab54518 in S36a mutant samples. This code will give you 1 freeprimary antibodybefore the expiration date. To redeem this offer, please submit an Abreview for S36a mutants and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: https://www.abcam.com/Abreviews.

Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

Read More

Answer

Thank you for your email. For activated SHC (66 kDa isoform, phosphorylation on Ser36), we have only 1 antibody: ab54518. It has been tested in WB and with mouse. https://www.abcam.com/shc-phospho-s36-antibody-6e10-ab54518.html For non-activated SHC (66 kDa isoform), we have only antibody ab103134. https://www.abcam.com/SHC-antibody-ab103134.html Since the immunogen was selected from the N-terminal region (aa 72-101) which is missing in the 52 and 46 kDa isoforms, the antibody will recognize only the 66 kDa isoform. Thus, the antibody is specific to the 66 kDa isoform. However, if the protein is phosphorylated at Ser36, this antibody will detect the phospho-protein as well. The antibody would basically measure total SHC protein (activated AND unactivated SHC 66 kDa). Also, the antibody has not yet been tested in mouse, only in human and WB, IHC-P. The homology between human and mouse is only 73% for the immunogen sequence and thus, might work or might not work. It is difficult to predict. Ideally, you want to use an antibody raised against a region within aa 1-60 as the homology is much higher (close to 100%). Unfortunately, we do not carry such antibody. If you decide to try ab103134 in mouse, I'd be happy to offer you our testing discount. For details please below: For UNTESTED species and/or applications, we have established a testing discount program. Here is a brief description of how it works: The testing discount program is for customers who like to use an antibody/protein on an untested species/application. You would purchase the antibody at full price, test it and submit an Abreview with your data (positive or negative). On your next order you will receive a discount for ONE antibody at the full price (100%) of the antibody you have tested. The terms and conditions applicable to this offer can be found here: https://www.abcam.com/collaborationdiscount. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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