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:
See all 16 Publications for this product

Customer reviews and Q&As

1-10 of 17 Abreviews or Q&A

Answer

We do share your concerns regarding mouse on mouse staining of kidney tissues. At Abcam we recommend following the attached mouse on mouse protocol tips to reduce background from this type of staining. While we do not have data regarding kidney tissue staining, we have shown this product to be effective in ICC on HepG2 (liver hepatocellular carcinoma) using a traditional staining procedure which includes 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween to permeabilise the cells and block non-specific protein-protein interactions. However if you wish to remove detergents from your experiment I can suggest fixation with Methanol or Acetone (both of which fix and permeablize) as an alternative to a formalin fix/ detergent permeablization.

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Abcam has not validated the combination of species/application used in this Abreview.
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

Question

thank you for so fast reply!
1. "the clone of the different H1 antibody" - ab71594 anti-Histone H1 [AE-4];
2. the anti-PIP2 mouse monoclonal IgM antibody (cat. no.: ab11039; lot: GR
108137-1;
> Questionnaire:
> Order Details
> Antibody code:
> ab11039
> Lot number:
> GR 108137-1
> Purchase order number
> or preferably Abcam order number:
1204561
> General Information
> Antibody storage conditions (temperature/reconstitution etc)
> the storage conditions were exactly those recommended in the
DataSheet - storage at +4 C short term (less than 1
week), then probing for immunofluorescence
>
> Description of the problem (high background, low signal, non-specific
> satining etc.)
no labeling at all
>
> Sample (Species/Tissue/Cell Type/Cell Line etc.)
> HeLa mono layer cell culture
>
> Fixation of sample
> (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)
> 3% Paraformaldehyde in PBS, 20 min, RT
>
> Antigen retrieval (Enzymatic method, Heat mediated technique etc.)
> None
>
> Permeabilization step
> (simultaneously with fixation) 0.1% Triton X-100 in PBS, 20 min, RT
>
> Blocking conditions (Buffer/time period, Blocking agent etc.)
> 5% normal goat serum in PBS, 20 min, RT
>
> Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time,
> Wash step)
Abcam the anti-PIP2 mouse monoclonal IgM antibody (cat. no.:
ab11039; lot: GR 108137-1), probed 1:50-20 µg/ml and
1:100-10 µg/ml in PBS, incubation in moist chamber during 1 h RT,
then washing three times by 5 min each in PBST
> > Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation
time, Wash step)
goat anti-mouse coupled with Alexa 555, diluted 1:400 in PBST,
incubation in moist chamber in darkness during 1 h
RT, then washing three times by 5 min each in PBST and 5 min in PBS
>
> Detection method
Fluorescent microscopy, conditions of acquisition: Intensity – 3;
Gain – 1; Exposure – 0.23 sec.
>
> Positive and negative controls used (please specify)
> as a positive control, well-working primary antibody of the same
species was used followed by the same secondary
antibody
in a negative control, primary antibody was omitted, and the cells
were incubated parallely in the diluent (PBS)
under the same conditions during the same time
>
Optimization attempts (problem solving)
> How many times have you tried the IHC?
> the immunolabeling experiment was repeated three times
>
> Have you run a "No Primary" control?
> Yes
>
> Do you obtain the same results every time?
> Yes
>
>
> What steps have you altered?
we probed two different concentrations of the antibody each time,
but the results were always negative
>
> Additional Notes
> We suppose this batch is not good for immunofluorescence. We
really need the antibody against PIP2 for our
experiments. May we suggest that you send us new batch of the
ab11039 antibody (2 tubes by 100 µg each)?
>
> We would appreciate if you are also able to provide and image which
would help us to assess the results
the images are attached to this letter.
3. the anti-Histone H1 mouse monoclonal IgG2a antibody (cat.no.: ab71594;
lot: GR63952-4)
> Questionnaire:
> Order Details
> Antibody code:
> ab71594
> Lot number:
> GR63952-4
> Purchase order number
> or preferably Abcam order number:
1204550
> General Information
> Antibody storage conditions (temperature/reconstitution etc)
> the storage conditions were exactly those recommended in the
DataSheet - shipped at +4 C, then immediately probing for
immunofluorescence
>
> Description of the problem (high background, low signal, non-specific
> satining etc.)
unspecific pattern in nucleoplasm
>
> Sample (Species/Tissue/Cell Type/Cell Line etc.)
> HeLa mono layer cell culture
>
> Fixation of sample
> (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)
> 3% Paraformaldehyde in PBS, 20 min, RT
>
> Antigen retrieval (Enzymatic method, Heat mediated technique etc.)
> None
>
> Permeabilization step
> (simultaneously with fixation) 0.1% Triton X-100 in PBS, 20 min, RT
>
> Blocking conditions (Buffer/time period, Blocking agent etc.)
> 5% normal goat serum in PBS, 20 min, RT
>
> Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time,
> Wash step)
Abcam the anti-Histone H1 mouse monoclonal IgG2a antibody
(cat.no.: ab71594; lot: GR63952-4), probed 1:50-20 µg/ml and
1:100-10 µg/ml in PBS, incubation in moist chamber during 1 h RT,
then washing three times by 5 min each in PBST
> > Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation
time, Wash step)
goat anti-mouse coupled with Alexa 555, diluted 1:400 in PBST,
incubation in moist chamber in darkness during 1 h
RT, then washing three times by 5 min each in PBST and 5 min in PBS
>
> Detection method
Fluorescent microscopy, conditions of acquisition: Intensity – 3;
Gain – 1; Exposure – 0.23 sec.
>
> Positive and negative controls used (please specify)
> as a positive control, well-working primary antibody of the same
species was used followed by the same secondary
antibody
in a negative control, primary antibody was omitted, and the cells
were incubated parallely in the diluent (PBS)
under the same conditions during the same time
>
Optimization attempts (problem solving)
> How many times have you tried the IHC?
> the immunolabeling experiment was repeated three times
>
> Have you run a "No Primary" control?
> Yes
>
> Do you obtain the same results every time?
> Yes
>
>
> What steps have you altered?
we probed two different concentrations of the antibody each time,
but the results were always negative
>
> Additional Notes
> We suppose this batch is not good for immunofluorescence. We
really need the antibody against Histone H1 for our
experiments. May we suggest that you send us new batch of the
ab71594 antibody (1 tube by 50 µg)?
> We would appreciate if you are also able to provide and image which
would help us to assess the results
the images are attached to this letter.
Thank you very much in advance!
Sincerely Yours,

Read More
Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and also gives us vital information for our monitoring of product quality

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. I would like to reassure you that this antibodies are tested and covered by our guarantee for ICC/IF and human samples.

Before deciding how to proceed with this particular case, I would like to offer some suggestions to help optimise the results. I would also appreciate if you can confirm some details of the procedure.

1. PIP2 antibody:

a) Because the PIP2 protein is a membrane protein it is very important to permeabalise carefully. We would recommend to permeableise with 0.1% Tween instead of Triton and use the following protocol, which can be found on the datasheet:

- cells were 4% PFA fixed (10 min)

- 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

- cells were then incubated with the antibody (ab11039, 5 µg/ml) overnight at +4°C.

b) could you kindly confirm if the same secondary antibody was used for the ab11039 and the ab71594? These two antibodies contain a different Isotype, which is IgM for the PIP2 antibody and IgG2a for the Histon H1 antibody. If the secondary antibody detects an IgG isotype we would not expect it to detect the IgM as well.

2. Histon H1:

a) The Histon H1 is a nuclear protein and there are not many suggestions relating the protocol to make. However, different antibodies need different optimisation steps. Even so a protocol works with one antibody, another antibody against the same target might need an optimised protocol. We like to suggest to try an incubation with the recommended dilution of 5ug/ml for the ab71594.

b) Could you please confirm if this antibody was stored in aliqouts at - 20 degrees directly after arrival?

We are happy to offer this technical support. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

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Question

Dear Sir or Madam,
We purchased the anti-PIP2 mouse monoclonal IgM antibody (cat. no.:
ab11039; lot: GR 108137-1; 2 tubes by 100 µg each) and the anti-Histone H1
mouse monoclonal IgG2a antibody (cat.no.: ab71594; lot: GR63952-4; 1 tube
by 50 µg). We probed these antibodies in immunofluorescent study.
Unfortunately, the antibodies did not react properly with the
corresponding antigens:
- there was no labeling at all for the anti-PIP2 mouse monoclonal IgM
antibody (cat. no.: ab11039) and
- there was unspecific pattern in nucleoplasm for the anti-Histone H1
mouse monoclonal IgG2a antibody (cat.no.: ab71594).
The fluorescent microscopic images that confirm these negative result are
attached to this letter.
For the anti-PIP2 antibody, in the left column, a negative control image
is shown with the same secondary antibody. In the right column, the image
of cell incubated with the anti-PIP2 mouse monoclonal IgM antibody (cat.
no.: ab11039) is shown.
For the anti-Histone H1 antibody, in the left column, a normal
immunofluorescent pattern for the antigen is shown with a different
antibody. In the right column, the image of cell incubated with the
anti-Histone H1 mouse monoclonal IgG2a antibody (cat.no.: ab71594) is
shown.
The incubations were performed in parallel using the same protocol, and
the imaging conditions were identical. This experiment was repeated
several times, a representative picture is shown.
We suppose these batches are not good for immunofluorescence. We really
need the antibodies against PIP2 and Histone H1 for our experiments. May
we suggest that you send us new batches of the ab11039 antibody (2 tubes
by 100 µg each) and ab71594 antibody (1 tube by 50 µg)?
Thanking you in advance for your hopefully positive reply,
Sincerely Yours,

Read More
Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from these antibodies.

I would like to reassure you that this antibodies are tested and covered by our 6 month guarantee for ICC/IF and human samples (ab11039 also for mouse and rat samples). In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily.

I would appreciate if you could also confirm the clone of the different H1 antibody.

Thank you for your time and cooperation. We look forward to receiving the completed questionnaire.



Questionnaire:

Order Details
Antibody code:

Lot number:

Purchase order number
or preferably Abcam order number:


General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, low signal, non-specific satining etc.)

Sample (Species/Tissue/Cell Type/Cell Line etc.)


Fixation of sample (Ethanol/Methanol/Acetone/Paraformaldehyde/Other/Duration etc.)


Antigen retrieval (Enzymatic method, Heat mediated technique etc.)


Permeabilization step


Blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method


Positive and negative controls used (please specify)


Optimization attempts (problem solving)
How many times have you tried the IHC?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No


What steps have you altered?


Additional Notes


We would appreciate if you are also able to provide and image which woudl help us to assess the results

Read More

Answer

Thank you for contacting us.

I personally don't have experience with using a freeze-thaw permeabilization, but from doing a quick literature search it does seem to be a good method for staining intracellular membrane proteins.

http://www.ncbi.nlm.nih.gov/pubmed/12667681

I can't whole-heartedly recommend the freeze-thaw perm to be used with this antibody because I can't find any references citing the use. For ICC/IF, these are the protocols that were used:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3042415/pdf/1471-2121-12-4.pdf

Cells were washed once with PBS (pH 7.4), fixed in 4% formaldehyde/0.05% glutaraldehyde for 5 min at room temperature (RT), permeabilized with 10 μg/ml digitonin (Sigma Aldrich, Munich/Germany) or with 0.5% Triton X-100 for 6 min (Sharma et al., 2008), and then blocked with 1% BSA for 20 min. Cells were then incubated with a primary antibody overnight at 4°C. After washing with PBS, cells were further treated with fluorescence-coupled antibodies (FITC/TexasRed) for 1 h at RT. Dabco-glycerin in PBS was used as a mounting solution. The sources of the antibodies included mouse monoclonal anti-Na+/K+-ATPase (a3 subunit, 1:200, Sigma Aldrich, Munich/Germany); goat polyclonal anti-phospho Na+/K+-ATPase (a-Ser 943, 1:100, Santa Cruz Biotechnology Inc., Heidelberg/Germany); mouse monoclonal anti-NHE-3 isoform (1:50, BD Transduction Labs, Heidelberg/Germany); mouse monoclonal anti-phospho NHE-3 (Ser 552, 1:500, Novus Biologicals, Heford/Germany); rabbit anti-human vinculin (1:200, Sigma Aldrich, Munich/Germany); mouse monoclonal anti-phosphatidylinositol 4,5-bisphosphate (PIP2, 1:200, Abcam, Cambridge/UK); rabbit polyclonal anti-beta actin (1:1000, Novus Biologicals, Heford/Germany); fluorescein-isothiocyanate (FITC)-coupled anti-mouse, anti-rabbit, or antigoat (1:500, Dianova, Hamburg/Germany); Texas Redcoupled anti-mouse and anti-rabbit (1:500, Dianova, Hamburg/Germany).

http://www.jbc.org/content/284/8/4836.full.pdf+html

Briefly, for PIP2 staining cells were fixed on ice for 3 h in 4% paraformaldehyde-Dulbecco’s modified Eagle’s medium with 2mM EGTA, washed three times in 50 mM NH4Cl-phosphate buffered saline, and permeabilized for 4 h at 4 °C in permeabilization buffer (1 mM MgCl2, 0.2% saponin, 1% fetal calf serum, 0.1% bovine serum albumin, 50 mM glycine, 0.05% NaN3 in phosphate-buffered saline). Incubation with anti-PIP2 antibody (1/50) in permeabilization buffer was performed overnight at 4 °C and then with the secondary antibody for 2 h at 4 °C.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you very much for your reply.
Since PIP2 seems to be ubiquitously expressed, I would expect most, if not all, cells to be positive controls. For ab11039, HepG2 may be the best positive control since the antibody has been tested in this cell line. I'm not sure of a good negative control, besides knockout samples or siRNA knockdowns. You could also overexpress PIP2 and compare the staining to wild type cells.
I am sorry that I can't be more help in this regard, but if you need anything else from me please don't hesitate to ask. Have a nice day!

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Answer

Thank you again for your reply and for your patience.
I have followed-up with the lab and the secondary antibody mentioned in the image caption was actually an anti-mouse IgM, not IgG. We have corrected this in the caption. Thank you very much for pointing out this error.
Please let me know if you have any further questions and I'll be happy to help. Have a nice day.

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Answer

Thank you very much for your reply.
I'll need to check with the lab to confirm the type of secondary antibody that was used in this ICC/IF protocol. The anti-mouse IgG secondary antibody described in the image caption would still probably cross-react with an IgM primary, as this secondary is specific for both the heavy and light chains conserved across isotypes. I'll get back to you once I have more information from the lab.
I looked through the literature but unfortunately couldn't find any images showing the localization of PIP2 in Schwann cells, so I'm not sure if the staining should be more membranous. To detect the membrane PIP2, I recommend fixing with 4% PFA followed by brief permeabilization with Tween20, digitonin, or saponin. I don't suggest using Triton as high concentrations or long incubations with Triton can dissolve the plasma membrane and inhibit the staining of membranous PIP2.
Please let me know if you have any questions, and I will be back in touch once I've heard from the lab about the secondary antibody used in the lab on the datasheet.

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Answer

Thank you very much for contacting us with your question.
I've looked through some literature, and it looks like PIP2 can be found in the nucleus as well as on the plasma membrane-
http://jcs.biologists.org/content/114/13/2501.long
http://www.ncbi.nlm.nih.gov/pubmed/8312134
http://www.jbc.org/content/284/8/4836.long
In the top article above, the authors found that the PIP2 can associate with particles in the nucleus that resemble interchromatin granule clusters, which would correlate with the clusters seen in the ICC/IF image on the datasheet. It is possible that different cell types or experimental conditions will affect the exact localization of the PIP2 staining. Is there a specific cell line or treatment that you're working with? I'd be happy to look through the literature to see if there is any information regarding where PIP2 might localize in your samples.
I hope that this information will be useful, but if you have any further questions or need anything else, please let me know and I'll be happy to help.

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Question
Answer

Thank you for your call today and for letting us know about the trouble with this antibody.

Your credit note ID is ***

I am sorry that this antibody did not perform as stated on the datasheet. If payment has already been made on the original order and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. Our accounting department can be contacted by email at us.credits@abcam.com orat 888-772-2226.Please refer to the credit note ID in any correspondence with our accounting department.

Please let me know if you have further questions or if there is anything else that we can do for you, and I'll be happy to help.

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1-10 of 17 Abreviews or Q&A

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