Anti-PIP2 抗体 [2C11] (ab11039)

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.

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.

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

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.

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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!

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.

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.

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.