Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-GRB2 antibody [Y301] (ab32111)

Reviews (1)Q&A (2)References (1)
Western blot - Anti-GRB2 antibody [Y301] (ab32111)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRB2 antibody [Y301] (ab32111)
  • Immunocytochemistry/ Immunofluorescence - Anti-GRB2 antibody [Y301] (ab32111)
  • Flow Cytometry - Anti-GRB2 antibody [Y301] (ab32111)
  • Western blot - Anti-GRB2 antibody [Y301] (ab32111)
  • Anti-GRB2 antibody [Y301] (ab32111)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [Y301] to GRB2
  • Suitable for: WB, IHC-P, ICC/IF, Flow Cyt
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Overview

  • Product name

    Anti-GRB2 antibody [Y301]
    See all GRB2 primary antibodies

  • Description

    Rabbit monoclonal [Y301] to GRB2

  • Host species

    Rabbit

  • Specificity

    ab32111 recognises GRB2. This antibody can also detect the splice isoform GRB3-3 of GRB2.

  • Tested Applications & Species

    Application Species
    Flow Cyt
    Human
    ICC/IF
    Rat
    IHC-P
    Human
    WB
    Mouse
    Rat
    Human
    See all applications and species data

  • Immunogen

    Synthetic peptide within Human GRB2 aa 1-100 (N terminal). The exact sequence is proprietary.

  • Positive control

    • Human breast carcinoma, PC12 cells WB: HCT116, HeLa, PC-12, NIH/3T3, HEK-293, RAW 264.7 and C6 cell lysates

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

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

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.

    One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.

    Learn more here.

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab32111 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

Application Species
Flow Cyt
Human
ICC/IF
Rat
IHC-P
Human
WB
Mouse
Rat
Human
Application Abreviews Notes
WB (1)
1/1000 - 1/5000. Predicted molecular weight: 25 kDa.
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF
1/100 - 1/250.
Flow Cyt
1/40.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
Notes
WB
1/1000 - 1/5000. Predicted molecular weight: 25 kDa.
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF
1/100 - 1/250.
Flow Cyt
1/40.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
  • Application notes
    Is unsuitable for IP.

    • Target

    • Function

      Adapter protein that provides a critical link between cell surface growth factor receptors and the Ras signaling pathway.
      Isoform GRB3-3 does not bind to phosphorylated epidermal growth factor receptor (EGFR) but inhibits EGF-induced transactivation of a RAS-responsive element. Isoform GRB3-3 acts as a dominant negative protein over GRB2 and by suppressing proliferative signals, may trigger active programmed cell death.

    • Sequence similarities

      Belongs to the GRB2/sem-5/DRK family.
      Contains 1 SH2 domain.
      Contains 2 SH3 domains.

    • Domain

      The SH3 domains mediate interaction with RALGPS1 and SHB.

    • Cellular localization

      Golgi apparatus.
    • Information by UniProt

    • Database links

      see all

    • Alternative names

      • Abundant SRC homology antibody
      • Adapter protein GRB2 antibody
      • ASH antibody
      • Ash protein antibody
      • EGFRBP GRB2 antibody
      • Epidermal growth factor receptor binding protein antibody
      • Epidermal growth factor receptor binding protein GRB2 antibody
      • GRB 2 antibody
      • GRB2 adapter protein antibody
      • Grb2 antibody
      • GRB2_HUMAN antibody
      • Grb3 3 antibody
      • Growth factor receptor bound protein 2 antibody
      • Growth factor receptor bound protein 3 antibody
      • Growth factor receptor-bound protein 2 antibody
      • HT027 antibody
      • MST084 antibody
      • MSTP084 antibody
      • NCKAP2 antibody
      • OTTHUMP00000166096 antibody
      • OTTHUMP00000166097 antibody
      • OTTHUMP00000166098 antibody
      • Protein ash antibody
      • SEM5 antibody
      • SH2/SH3 adapter GRB2 antibody
      see all

    Images

    • Western blot - Anti-GRB2 antibody [Y301] (ab32111)
      Western blot - Anti-GRB2 antibody [Y301] (ab32111)
      All lanes : Anti-GRB2 antibody [Y301] (ab32111) at 1/2000 dilution

      Lane 1 : Wild-type HCT116 cell lysate
      Lane 2 : GRB2 knockout HCT116 cell lysate
      Lane 3 : HeLa cell lysate
      Lane 4 : HEK293 cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Predicted band size: 25 kDa
      Observed band size: 25 kDa



      Lanes 1 - 4: Merged signal (red and green). Green - ab32111 observed at 25 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

      ab32111 was shown to react with GRB2 in HCT 116 wild-type cells in western blot with loss of signal observed in GRB2 knockout cell line ab273715 (GRB2 knockout cell lysate ab275248). HCT 116 wild-type and GRB2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab32111 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRB2 antibody [Y301] (ab32111)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GRB2 antibody [Y301] (ab32111)

      Immunohistochemical analysis of GRB2 expression in paraffin embedded human breast carcinoma tissue section, using 1/100 ab32111. Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

    • Immunocytochemistry/ Immunofluorescence - Anti-GRB2 antibody [Y301] (ab32111)
      Immunocytochemistry/ Immunofluorescence - Anti-GRB2 antibody [Y301] (ab32111)
      Immunofluorescent analysis of GRB2 expression in PC12 cells, using 1/100 ab32111.
    • Flow Cytometry - Anti-GRB2 antibody [Y301] (ab32111)
      Flow Cytometry - Anti-GRB2 antibody [Y301] (ab32111)
      Overlay histogram showing SH-SY5Y cells stained with ab32111 (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 (ab32111, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

    • Western blot - Anti-GRB2 antibody [Y301] (ab32111)
      Western blot - Anti-GRB2 antibody [Y301] (ab32111)
      All lanes : Anti-GRB2 antibody [Y301] (ab32111) at 1/5000 dilution (purified)

      Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
      Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates
      Lane 3 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
      Lane 4 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysates
      Lane 5 : RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates
      Lane 6 : C6 (Rat glial tumor glial cell) whole cell lysates

      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: 25 kDa
      Observed band size: 25 kDa



      Blocking/Diluting Buffer and concentration: 5% NFDM/TBST

      We are unsure how to define the extra bands.

    • Anti-GRB2 antibody [Y301] (ab32111)
      Anti-GRB2 antibody [Y301] (ab32111)

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    References (1)

    Publishing research using ab32111? Please let us know so that we can cite the reference in this datasheet.

    ab32111 has been referenced in 1 publication.

    • Parodi B  et al. Fumarates modulate microglia activation through a novel HCAR2 signaling pathway and rescue synaptic dysregulation in inflamed CNS. Acta Neuropathol 130:279-95 (2015). WB . PubMed: 25920452

    Customer reviews and Q&As

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    1-3 of 3 Abreviews or Q&A

    Question

    What is the recommended antigen retrieval buffer for these three antibodies?

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    Answer

    Thank you for contacting us.

    The following standard heat-mediated citrate buffer antigen retrieval was used for the IHC validation of ab32111 and ab111031. Antibody ab111586 used citrate buffer in a pressure cooker at 120C for 5 minutes but a steamer or waterbath at 95-100C will be an acceptable substitute.

    Place slides in a container of 0.01 M sodium citrate buffer, pH 6.0 heated to 100°C and maintain this temperature for 20 minutes by placing the container in a steamer or heated waterbath. After 20 minutes, remove from heat and let stand at room temperature in buffer for 20 minutes. Rinse in 1X TBS with Tween (TBST) for 1 minute at RT.

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

    Read More

    Question

    What was the antigen retrieval method that was used to generate the picture on your datasheet?

    Read More
    Answer

    We used a heat mediated antigen retrieval method with sodium citrate, pH. 6.0.

    Read More

    Western blot abreview for Anti-GRB2 antibody [Y301]

     Good
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Raji)
    Loading amount
    40 µg
    Specification
    Raji
    Gel Running Conditions
    Reduced Denaturing (12%)
    Blocking step
    Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
    Read More

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    Verified customer

    Submitted Sep 11 2009

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