Recombinant
RabMAb

Recombinant Anti-SRC Family (phospho Y418) antibody [EP503Y] (ab40660)

Overview

  • Product name

    Anti-SRC Family (phospho Y418) antibody [EP503Y]
    See all SRC Family primary antibodies
  • Description

    Rabbit monoclonal [EP503Y] to SRC Family (phospho Y418)
  • Host species

    Rabbit
  • Specificity

    ab40660 recognises Lyn phosphorylation on Tyrosine 396. BLAST analysis show 100% homology with Hck (pY410) and Lck (pY393), and 92% homology with Src (pY418), Fyn (pY419) and Yes (pY425) Therefore, the antibody may cross-react with these proteins.
  • Tested applications

    Suitable for: WB, Flow Cyt, ICC/IF, ELISAmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human SRC Family aa 350-450 (phospho Y418). The exact sequence is proprietary.
    Database link: P07948
    (Peptide available as ab202645)

  • Positive control

    • WB: HeLa and C6 cell lysates treated with pervanadate. ICC/IF: HeLa cells treated with pervanadate. Flow Cyt: HeLa cells treated with pervanadate.
  • General notes

    This protein is known to be similar in amino acid sequence to HCK (P08631), LCK (P06239), FYN (P06241), YES1 (P07947), and SRC (P12931). Therefore, cross-reactivity with these homologous proteins may be observed. We would be happy to provide immunogen alignment information upon request.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 50% Glycerol, 0.05% BSA, 49% PBS
  • Concentration information loading...
  • Clonality

    Monoclonal
  • Clone number

    EP503Y
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab40660 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 - 1/10000. Detects a band of approximately 56 kDa (predicted molecular weight: 61 kDa).Can be blocked with Lyn (phospho Y396) peptide (ab202645).

Induction of cells with Pervanadate: Prepare 500mM stock Sodium Orthovanadate in PBS. To make 100mM (100x) Pervanadate Na3VO4 right before experiment, add 400ul 500mM Sodium Orthovanadate to 1560ul H2O and then 40ul H2O2, vortex and incubate at room RT for 5-10min. Cells are serum-starved overnight, and then treated with freshly made Pervanadate (100x, final concentration 1mM) for 20min at 37°C.

Flow Cyt 1/50.

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

ICC/IF Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.

Target

  • Relevance

    SRC Family Negative Regulatory Phosphorylation Site belongs to the protein kinase superfamily, Tyr protein kinase family, SRC subfamily. It contains one protein kinase domain, one SH2 domain and one SH3 domain. It may serve as part of a signaling pathway coupling the Fc receptor to the activation of the respiratory burst and may also contribute to neutrophil migration and may regulate the degranulation process of neutrophils. SRC Family Negative Regulatory Phosphorylation Site is expressed predominantly in cells of the myeloid and B lymphoid lineages. There are two named isoforms.
  • Cellular localization

    Isoform 1: Membrane; Lipid anchor. Isoform 2: Membrane; Lipid anchor. Cytoplasm.
  • Database links

  • Alternative names

    • ASV antibody
    • Protooncogene SRC, Rous sarcoma antibody
    • SRC antibody
    • SRC1 antibody

Images

  • All lanes : Anti-SRC Family (phospho Y418) antibody [EP503Y] (ab40660) at 1/2000 dilution (purified)

    Lane 1 : Untreated HeLa whole cell lysate
    Lane 2 : Pervanadate treated HeLa whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 61 kDa
    Observed band size: 56-59 kDa
    why is the actual band size different from the predicted?


    Exposure time: 3 seconds


    Blocking and dilution buffer: 5% BSA/TBST.

  • All lanes : Anti-SRC Family (phospho Y418) antibody [EP503Y] (ab40660) at 1/1000 dilution (purified)

    Lane 1 : Untreated C6 whole cell lysate
    Lane 2 : Pervanadate treated C6 whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 61 kDa
    Observed band size: 56-59 kDa why is the actual band size different from the predicted?


    Exposure time: 1 second


    Blocking and dilution buffer: 5% BSA/TBST.

  • All lanes : Anti-SRC Family (phospho Y418) antibody [EP503Y] (ab40660) at 1/1000 dilution (purified)

    Lane 1 : Pervanadate treated Jurkat cell lysate with Lyn non-phospho peptide
    Lane 2 : Pervanadate treated Jurkat cell lysate with Lyn (pY396) phospho peptide
    Lane 3 : Pervanadate treated Jurkat cell lysate with Lck non-phospho peptide
    Lane 4 : Pervanadate treated Jurkat cell lysate with Lck (pY393) phospho peptide
    Lane 5 : Pervanadate treated Jurkat cell lysate with Src non-phospho peptide
    Lane 6 : Pervanadate treated Jurkat cell lysate with Src (pY418) phospho peptide

    Secondary
    All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Predicted band size: 61 kDa
    Observed band size: 56-59 kDa why is the actual band size different from the predicted?


    Exposure time: 3 minutes


    Blocking and dilution buffer: 5% NFDM/TBST.

  • Immunocytochemistry/Immunofluorescence analysis of HeLa cells (untreated and treated with 1mM pervanadate for 15 minutes) labelling SRC Family (phospho Y418) with purified ab40660 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

    Control 1: primary antibody (1/250) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).

    Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000).

  • ICC/IF image of unpurified ab40660 stained PC12 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab40660, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Flow Cytometry analysis of HeLa cells (treated with 1mM pervanadate for 30 minutes) labelling SRC Family (phospho Y418) with purified ab40660 at 1/80 (red). Cells were fixed with 4% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody.

    Black - Isotype control, rabbit monoclonal IgG with pervanade treated HeLa cells.

    Blue - Unlabelled control, pervanadate treated HeLa cells without incubation with primary and secondary antibodies.

    Red - Pervandate treated HeLa cells with ab40660.

    Green - Untreated HeLa cells labelled with ab40660.

  • Direct ELISA antibody dose-response curve using unpurified ab40660 at 0-1000 ng/ml. Antigen concentration of 1000 ng/mL. An alkaline phosphatase-conjugated goat anti-rabbit IgG (H+L) (1/2500) was used as the secondary antibody.

References

This product has been referenced in:

  • Hong X & Yu JJ Silencing of lysyl oxidase-like 2 inhibits the migration, invasion and epithelial-to-mesenchymal transition of renal cell carcinoma cells through the Src/FAK signaling pathway. Int J Oncol 54:1676-1690 (2019). Read more (PubMed: 30816490) »
  • Nie Y  et al. A novel small inhibitor, LLL12, targets STAT3 in non-small cell lung cancer in vitro and in vivo. Oncol Lett 16:5349-5354 (2018). Read more (PubMed: 30250605) »
See all 24 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - whole cell (U2OS)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
U2OS
Blocking step
Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted May 30 2018

Application
Western blot
Loading amount
60 µg
Gel Running Conditions
Reduced Denaturing (10% PAGE Gel)
Sample
Human Cell lysate - whole cell (Hela cells)
Specification
Hela cells
Treatment
100ng/ml IL6 for 15 minutes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Feb 12 2015

Answer

The difference between lyn a and lyn b is that lyn b does not contain amino acids 23-43 of lyn a. Since this antibody recognises a region around tyrosine 396, ab40660 can detect both lyn a and lyn b therefore it is very probable that this doublet band seen at around 57 kDa corresponds to a band at 56 kDa for lyn b and 59 kDa for lyn a.

Read More

Answer

Thank you for your enquiry regarding ab40660.
Based on the sequence homology, it is unlikely that this antibody would cross react with the other tyrosines of Lyn. However, it might cross-react with LCK, HCK and possibly Src when phosphorylated at equivalent sites since Lyn shares high sequence homology with other Src family members.
Please note that the development of all phospho-specific antibodies requires to screen on phospho specific peptide and counter-screen on its corresponding non-phospho specific control peptide.
If you need any further assistance in the future, please do not hesitate to contact me.

Read More

Answer

ab33906 lot YH090207C - 0.3260 mg/ml

ab33862 lot YH082410D - 0.6380 mg/ml

ab32429 lot YG080503 - 0.0790 mg/ml

ab40660 lot YI070802CS - 0.1030 mg/ml

ab32445 lot YI072804DS - 0.0250 mg/ml

Read More

Answer

Thank you for your enquiry.

I can confirm that we have a dedicated team at Abcam who review the literature in order to find publications that have used our products. In this case, the following publication was found, showing use of this antibody in mouse. This link is available on the datasheet:

Kim SW et al. Rational combined targeting of phosphodiesterase 4B and SYK in DLBCL. Blood 113:6153-60 (2009). WB; Mouse. PubMed: 19369227

Therefore, mouse has been added to the tested species list for ab40660 Lyn (phospho Y396) antibody [EP503Y].

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

Read More

Answer

Thank you for your enquiry.

These 2 antibodies have lot specific concentrations. If you could please forward me the lot numbers for ab40660 and ab33914 I'd be happy to enquire about the concentrations.

Thanks for the additional information!

Read More

Answer

Thank you for contacting Abcam.

We added the reactivity with mouse based on the following reference:

Kim SW et al. Rational combined targeting of phosphodiesterase 4B and SYK in DLBCL. Blood 113:6153-60 (2009).



The antibody is covered under our Abpromise for six months and is guaranteed to work in western blot onmouse samples . If you do experience any problems with it and we cannot resolve the issue, thenwe would be happy to either send a replacement antibody or to process a refund.

Please let me know if there is anything else I can help you with.

Read More

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