Recombinant
RabMAb

Recombinant Anti-SLP76 (phospho Y145) antibody [EP2853Y] (ab75829)

Overview

  • Product name

    Anti-SLP76 (phospho Y145) antibody [EP2853Y]
    See all SLP76 primary antibodies
  • Description

    Rabbit monoclonal [EP2853Y] to SLP76 (phospho Y145)
  • Host species

    Rabbit
  • Specificity

    This antibody detects SLP76 phosphorylated on tyrosine 145.
  • Tested applications

    Suitable for: ICC/IF, Dot blot, WB, Flow Cytmore details
    Unsuitable for: IHC-P
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide within Human SLP76 aa 100-200 (phospho Y145). The exact sequence is proprietary.
    Database link: Q13094

  • Positive control

    • Jurkat whole cell lysate (ab7899) treated with pervanadate.
  • General notes

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

     

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

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    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: 0.05% Sodium azide
    Constituents: 0.1% BSA, 40% Glycerol, 9.85% Tris glycine, 50% Tissue culture supernatant
  • Purity

    Tissue culture supernatant
  • Clonality

    Monoclonal
  • Clone number

    EP2853Y
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab75829 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 1/500.
Dot blot 1/1000.
WB 1/1000 - 1/10000. Predicted molecular weight: 60 kDa.
Flow Cyt 1/100.

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

  • Application notes
    Is unsuitable for IHC-P.
  • Target

    • Function

      Involved in T-cell antigen receptor mediated signaling.
    • Tissue specificity

      Highly expressed in spleen, thymus and peripheral blood leukocytes. Highly expressed also in T-cell and monocytic cell lines, expressed at lower level in B-cell lines. Not detected in fibroblast or neuroblastoma cell lines.
    • Sequence similarities

      Contains 1 SAM (sterile alpha motif) domain.
      Contains 1 SH2 domain.
    • Domain

      The SH2 domain mediates interaction with SHB.
    • Post-translational
      modifications

      Phosphorylated after T-cell receptor activation by ZAP-70.
    • Cellular localization

      Cytoplasm.
    • Information by UniProt
    • Database links

    • Alternative names

      • 76 kDa tyrosine phosphoprotein antibody
      • CG8697 antibody
      • LCP 2 antibody
      • LCP2 antibody
      • LCP2_HUMAN antibody
      • Lymphocyte cytosolic protein 2 antibody
      • SH2 domain containing leukocyte protein 76 KD antibody
      • SH2 domain containing leukocyte protein of 76kD antibody
      • SH2 domain containing leukocyte protein of 76kDa antibody
      • SH2 domain-containing leukocyte protein of 76 kDa antibody
      • SLP 76 antibody
      • SLP 76 tyrosine phosphoprotein antibody
      • SLP-76 antibody
      • SLP-76 tyrosine phosphoprotein antibody
      • SLP76 antibody
      • SLP76 tyrosine phosphoprotein antibody
      see all

    Images

    • All lanes : Anti-SLP76 (phospho Y145) antibody [EP2853Y] (ab75829) at 1/5000 dilution

      Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates
      Lane 2 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates. treated with pervanadate at 50nM for 5 min.
      Lane 3 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates. Treated with pervanadate at 50nM for 5 min. Then the membrane was incubated with phosphatase.

      Lysates/proteins at 15 µg per lane.

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

      Predicted band size: 60 kDa
      Observed band size: 76 kDa
      why is the actual band size different from the predicted?



      Blocking buffer 5% NFDM/TBST. Diluting buffer 5% NFDM/TBST

    • ab75829 staining SLP76 in Jurkat by Immunocytochemistry. Tissue was fixed with 4% paraformaldehyde. Samples were incubated with primary antibody (4.5 μg/ml). ab150077 AlexaFluor®488 Goat anti-Rabbit (1/1000) was used as the secondary antibody. Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 2.5 μg/ml and DAPI were used as counter stains.

      Confocal image showing the expression was increased after treatment with pervanadate (1mM, 30min) and then decreased after treatment with the Lambda Protein Phosphatase 31℃ for 2h

    • ab75829 staining SLP76 (phospho Y145) antibody in both treated (50mM of pervanadate for 5 minutes) and untreated Jurkat (human acute T cell leukemia) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000.

      DAPI was used as a nuclear counterstain.

    • All lanes : Anti-SLP76 (phospho Y145) antibody [EP2853Y] (ab75829) at 1/200000 dilution

      Lane 1 : Jurkat cell lysates un-treated
      Lane 2 : Jurkat cell lysates treated with pervanadate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

      Predicted band size: 60 kDa
      Observed band size: 76 kDa why is the actual band size different from the predicted?

    • Dot blot analysis of SLP76 (phospho Y145) phospho peptide (Lane 1), SLP76 Non-phospho peptide (Lane 2), labelling SLP76 (phospho Y145) with ab75829 at a dilution of 1/1000. Peroxidase conjugated goat anti-rabbit IgG (H+L)) was used as the secondary antibody at a dilution of 1/2500.

      Blocking and diluting buffer: 5% NFDM/TBST.

      Exposure time: 3 minutes.

    • Flow cytometric analysis of permeabilized Jurkat cells, untreated (green) or pervanadate treated (red) using ab75829 at a 1/100 dilution. Also, pervanadate Jurkat cells using the same antibody preincubated with phospho SLP76 peptide (blue) or non phospho SLP76 peptide (orange).

    References

    This product has been referenced in:

    • Wang QL  et al. T Cell Receptor (TCR)-Induced PLC-?1 Sumoylation via PIASxß and PIAS3 SUMO E3 Ligases Regulates the Microcluster Assembly and Physiological Function of PLC-?1. Front Immunol 10:314 (2019). Read more (PubMed: 30873169) »
    • Yang CY  et al. Dual-specificity phosphatase 14 (DUSP14/MKP6) negatively regulates TCR signaling by inhibiting TAB1 activation. J Immunol 192:1547-57 (2014). Read more (PubMed: 24403530) »
    See all 3 Publications for this product

    Customer reviews and Q&As

    1-4 of 4 Abreviews or Q&A

    Application
    Western blot
    Loading amount
    100000 cells
    Gel Running Conditions
    Reduced Denaturing (10%)
    Sample
    Human Cell lysate - whole cell (jurkats)
    Specification
    jurkats
    Treatment
    TCR activation with OKT3 and anti CD8
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

    Dr. Tejpal Gill

    Verified customer

    Submitted May 13 2014

    Answer

    Thank you for contacting us.

    Your credit note ID is 23976.

    I am sorry that this antibody did not perform as stated on the datasheet, I have asked our Finance department to issue a credit note for you. The credit note may be used in one of the following ways:

    (1) Redeemed against the original invoice if this hasn't already been paid.
    (2) Held on the account for use against a future order.
    (3) A full refund can be offered where no other invoices are outstanding.

    Please contact your Finance department to confirm how you would like the credit note to be used and ensure it is not redeemed without your knowledge.

    To specifically receive a refund please ask your Finance department to contact our Finance department at creditcontrol@abcam.com or by telephone using the information at the “Contact Us” link in the top right corner of our website.

    The credit note ID is for your reference only, please refer to the credit note ID in any correspondence with our accounting department. We will send you the completed credit note by email or postal mail with the actual credit note number which will start with the letters CGB.

    I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service should you require further expert advice

    Read More

    Question

    Here is the customer reply as attached files.
    Thank for your continuous help.


    1) Abcam product code ab75829

    2) Abcam order reference number: 6740/12

    3) Description of the problem: bands for p-SLP76 are not seen but only not specific bands

    4) Sample preparation:

    Type of sample (whole cell lysates, fraction, recombinant protein…): jurkat cell lysates.

    Lysis buffer: Tris HCl 50mM pH=7.5, NaCl 150mM, MgCl2 5mM, EDTA 2mM pH=7.5, triton 1%

    Protease inhibitors: protease inhibitor cocktail (P8340 sigma)

    Phosphatase inhibitors: phosphatase inhibitor cocktail (P5726 sigma)

    Reducing agent

    Boiling for ≥5 min? 5min

    Protein loaded 10ug/lane and 20ug/lane

    Positive control ---

    Negative control daudi and raji cell lysates

    5) Percentage of gel: lower- 10%, upper- 4%

    Type of membrane: cellulose nitrate membrane 0.20µm, 69X82mm

    Protein transfer verified

    Blocking agent and concentration: difco skim milk 5% in TBSX1

    Blocking time: 1hour

    Blocking temperature: room tem

    6) Primary antibody (If more than one was used, describe in “additional notes”): anti-SLP76 (phospho Y145) antibody EP2853Y

    Concentration or dilution: 1:500 in TBSTX1 and 5% BSA, 1:1000 in TBSTX1 and 5% BSA

    Diluent buffer

    Incubation time: over night

    Incubation temperature: 4Cº



    7) Secondary antibody: goat anti-rabbit IgG (H+L)-HRP conjugate

    Species:

    Reacts against:

    Concentration or dilution: 1:4000 in TBSX1

    Diluent buffer

    Incubation time: 1-1.5 hour

    Incubation temperature: room tem

    Fluorochrome or enzyme conjugate:



    8) Washing after primary and secondary antibodies:

    Buffer: TBSTX1

    Number of washes: 3 washes each one 7 min



    9) Detection method: EZ-ECL chemiluminescence detection kit for HRP



    10) How many times have you run this staining? twice

    Do you obtain the same results every time? yes

    What steps have you altered to try and optimize the use of this antibody? I used different concentration of the antibody and protein loading.

    Regards,

    Read More
    Answer

    Thank you for your enquiry regarding ab75829 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.

    I have discussed this case with the Lab. The protocol looks absolutely fine, the only recommendation we could make to dilute the antibody less if it is possible: 1/1000- 10,000.

    I am sorry for this inconvenience!

    Should your customer still has any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

    Read More

    Answer

    Thank you for your inquiry.

    The dilution factors for each application listed on the product datasheet for ab75829 are what we currently recommended, it is still relevant.

    If you wish me take this case further, could you please provide some further details of the protocol used and complete the following form (attached as a word document). It would be much appreciated if you could attach an image to the response.

    Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.

    Read More

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