Overview

  • Product name
    Anti-PIP2 antibody [2C11]
    See all PIP2 primary antibodies
  • Description
    Mouse monoclonal [2C11] to PIP2
  • Host species
    Mouse
  • Specificity
    Reacts with PtdIns(4,5)P2, Ptd(4)P and Ptd(3,4,5)P3.
  • Tested applications
    Suitable for: IHC-P, ICC/IF, ELISA, Neutralising, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Liposomes (prepared in PBS with lipid A, phosphatidylcholine (PC) and cholesterol) containing synthetic di-palmitoyl PtdIns(4,5)P2.

  • Positive control
    • IHC-P: FFPE human kidney normal and FFPE mouse normal brain. ICC: HepG2 cell line, Neuro2a cell line
  • General notes

    This antibody clone is manufactured by Abcam.

    For testing in lipid dot blot assay, follow the protocol used in Thomas et al. Biochem Soc Trans 27:648-52 (1999) (PMID: 10917659, please see the References tab).

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

Our Abpromise guarantee covers the use of ab11039 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
IHC-P Use a concentration of 1 - 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF Use a concentration of 1 - 5 µg/ml.
ELISA Use at an assay dependent concentration.
Neutralising Use at an assay dependent concentration.
IP Use at an assay dependent concentration. PubMed: 24214978

Target

  • Relevance
    Phosphatidylinositol 4,5-biphosphate (PIP2) is a membrane phospholipid that has been implicated in a variety of cellular processes, including synaptic vesicle recycling and signal transduction pathways. PLCD4 hydrolyzes PIP2 to generate 2 second messenger molecules diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3).

Images

  • ICC/IF image of ab11039 stained human HepG2 cells. The cells were 4% PFA 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 (ab11039, 5 µg/ml) overnight at +4°C.

    The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgM (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). This antibody also gave a positive IF result in HeLa, Hek293 and MCF7 cells.

  • IHC image of PIP2 staining in mouse normal brain formalin fixed paraffin embedded tissue section. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab11039, 1µg/ml overnight at +4°C. An HRP-conjugated secondary (Ab98679, 1/1000 dilution) was used for 1hr at room temperature. The section was counterstained with haematoxylin and mounted with DPX.

  • Immunofluorescence analysis of Human SaOS-2 (Human osteosarcoma) cells, staining PIP2 with ab11039. Cells were either unstimulated (upper panel) or stimulated with direct current (lower panel).

    Cells were fixed in formaldehyde, permeabilized and then blocked with 1% BSA for 20 min. Cells were then incubated with a primary antibody (1/200) overnight at 4°C. A FITC-conjugated anti-mouse IgG was used as the secondary antibody.
  • IHC image of PIP2 staining in a formalin fixed, paraffin embedded human normal kidney tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab11039, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

     

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

References

This product has been referenced in:
  • Hsieh CW & Yang WY Omegasome-proximal PtdIns(4,5)P2 couples F-actin mediated mitoaggregate disassembly with autophagosome formation during mitophagy. Nat Commun 10:969 (2019). Read more (PubMed: 30814505) »
  • Chen YH  et al. Asymmetric PI3K Activity in Lymphocytes Organized by a PI3K-Mediated Polarity Pathway. Cell Rep 22:860-868 (2018). ICC/IF . Read more (PubMed: 29420173) »
See all 17 Publications for this product

Customer reviews and Q&As

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1-4 of 4 Abreviews

Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293)
Loading amount
20 µg
Specification
HEK293
Gel Running Conditions
Non-reduced Denaturing (10% gel)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Feb 01 2013

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (24 hr EGF-treated LNCaP Androgen Dependent Prostat)
Specification
24 hr EGF-treated LNCaP Androgen Dependent Prostat
Fixative
Paraformaldehyde
Permeabilization
Yes - Triton X-100
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Skyla Carney

Verified customer

Submitted Oct 19 2011

Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (organ of Corti)
Specification
organ of Corti
Fixative
Paraformaldehyde
Permeabilization
No
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 20% · Temperature: 20°C

Dr. Vincent Michel

Verified customer

Submitted Dec 01 2010

Application
Western blot
Sample
Human Cell lysate - whole cell (Human lung epithelial cell)
Loading amount
50 µg
Specification
Human lung epithelial cell
Gel Running Conditions
Reduced Denaturing (12)
Blocking step
Milk as blocking agent for 18 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Jan 14 2009

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