Anti-ISWI (acetyl K753) antibody (ab10748)

This fast track antibody is not yet fully characterized. It is subject to these terms and conditions.

Overview

  • Product name
    Anti-ISWI (acetyl K753) antibody
  • Description
    Rabbit polyclonal to ISWI (acetyl K753)

    This product is a fast track antibody. It has been affinity purified and shows high titre values against the immunizing peptide by ELISA. Read the terms of use »

  • Host species
    Rabbit
  • Specificity
    The specificity of the antibody can be summarized as follows: 1. On Western blots probing recombinant ISWI which had been (or not) acetylated at K753 by GCN5, the antibody recognizes the acetylated ISWI with very high selectivity. No reaction with unacetylated ISWI has been observed. 2. Unexpectedly, there is a significant cross-reactivity with GCN5 under acetylation conditions (we assume autoacetylated GCN5). As shown in the figure, this cross-reactivity is low, since conditions can be found easily where the GCN5 band on Western blots is not observed. 3. In whole cell extracts the antibody cross-reacts with a number of small molecular weight bands. A faint signal at the position of ISWI is detected as well, which we think corresponds to ISWI since it is not present if ISWI is ablated using RNAi. The weakness of the signal may simply indicate that there is very little epitope present in these cells. In summary, we are confident that the antibody can be used on purified ISWI (e.g. ISWI immunoprecipitated from a cruder source).
  • Species reactivity
    Reacts with: Drosophila melanogaster, Sea urchin
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 700 - 800 of Fruit fly (Drosophila melanogaster) ISWI, acetylated at K753.

    Read Abcam's proprietary immunogen policy (Peptide available as ab16064.)

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    Preservative: 0.02% Sodium Azide
    Constituents: 1% BSA, PBS, pH 7.4
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Fast track antibodies constitute a diverse group of products that have been released to accelerate your research, but are not yet fully characterized. They have all been affinity purified and show high titre values against the immunizing peptide (by ELISA). Fast track terms of use

Application Abreviews Notes
ELISA Use at an assay dependent concentration. This antibody gave a positive result in ELISA against the immunizing peptide (ab16064). It gave a negative result in ELISA against the non-modified equivalent peptide (ab96245). This indicates that it is specific for the modified peptide.
ICC/IF Use at an assay dependent concentration. PubMed: 22949616
WB 1/500 - 1/2500. Detects a band of approximately 110 kDa (predicted molecular weight: 119 kDa).Can be blocked with D. melanogaster ISWI (acetyl K753) peptide (ab16064).

Target

  • Relevance
    ISWI is a component of the nucleosome remodeling factor complex (NURF), a protein complex that facilitates the perturbation of chromatin structure in vitro in an ATP-dependent manner. The hydrolysis of ATP during the remodeling of chromatin is likely to be mediated by ISWI, releasing inorganic phosphate. It is also a component of the ATP-utilizing chromatin assembly and remodeling factor (ACF) and of the chromatin accessibility complex (CHRAC). This subunit may serve as the energy-transducing component of chromatin-remodeling machines.
  • Cellular localization
    Nuclear
  • Database links
    • Alternative names
      • CHRAC 140 kDa subunit antibody
      • dISWI antibody
      • Imitation SWI antibody
      • NURF 140 antibody
      • NURF antibody
      • Protein imitation swi antibody
      see all

    Images

    This Fast-Track antibody is not yet fully characterised. These images represent inconclusive preliminary data.

    • Lanes 1-2 : Anti-ISWI (acetyl K753) antibody (ab10748) at 1/2500 dilution
      Lanes 3-4 : Anti-ISWI (acetyl K753) antibody (ab10748) at 1/500 dilution

      Lanes 2-3 : Recombinant ISWI (200 ng) was acetylated using 125ng of hGCN5 and a final concentration of 6 µM of 3H-AcCoA.

      Predicted band size: 119 kDa
      Observed band size: 120 kDa
      why is the actual band size different from the predicted?
      Additional bands at: 65 kDa. We are unsure as to the identity of these extra bands.

    • All lanes : Anti-ISWI (acetyl K753) antibody (ab10748) at 1/500 dilution

      Lane 1 : Drosophila melanogaster SL-2 cells treated for 8 days with ISWI dsRNAs
      Lane 2 : Drosophila melanogaster SL-2 cells treated for 8 days with control dsRNAs

      Lysates/proteins at 30 µg per lane.

      Predicted band size: 119 kDa
      Observed band size: 110 kDa why is the actual band size different from the predicted?
      Additional bands at: 55 kDa, 60 kDa, 65 kDa. We are unsure as to the identity of these extra bands.

    References

    This product has been referenced in:
    • L Black K  et al. Chromatin proteins and RNA are associated with DNA during all phases of mitosis. Cell Discov 2:16038 (2016). Read more (PubMed: 27807477) »
    • Yajima M  et al. ISWI contributes to ArsI insulator function in development of the sea urchin. Development 139:3613-22 (2012). ICC/IF ; Sea urchin . Read more (PubMed: 22949616) »
    See all 4 Publications for this product

    Customer reviews and Q&As

    1-8 of 8 Abreviews or Q&A

    Application
    Immunocytochemistry/ Immunofluorescence
    Blocking step
    0.1% Triton X-100, 0.1% BSA, 10% FCS, 0.1% sodium deoxycholate and 0.02% Thiomersal, an anti-fungal agent, in 1X PBS as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 24°C
    Sample
    Fruit fly (Drosophila melanogaster) Cell (Malpighian tubules, Salivary glands, Gut, etc)
    Specification
    Malpighian tubules, Salivary glands, Gut, etc
    Permeabilization
    Yes - 1% Triton X
    Fixative
    Paraformaldehyde

    Mr. Deo Prakash Deo Prakash

    Verified customer

    Submitted Jun 20 2013

    Answer

    Thank you for your email. I am sorry that I have not been clear enough yesterday. To receive the reward points it is necessary to submit an Abreview over the System. You will find the link to fill in all the data and attach the image on the product page as followed:

    https://www.abcam.com/iswi-acetyl-k753-antibody-ab10748.html

    Here you have to click on Abreview, that will lead to the following page:

    https://www.abcam.com/ISWI-acetyl-K753-antibody-ab10748-reviews.html

    On this page you will find a box that says:

    There are currently no Abreviews for ab10748.

    All products work in different combinations of applications, species and samples, so we encourage our customers to write a short Abreview, so that future customers can make an informed decision about the usefulness of a particular product for their specific experiments.

    Submit an Abreview and earn Abpoints

    I marked in red where you have to click to submit your data.

    The provided data will then automatically transferred to the Abcam Fast Track Team, who will review the details so that you can get the reward points.

    I hope this information is helpful to you. Please do not hesitate to contact me again if you have further questions.

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    Answer

    Thank you very much for your Email and this valuable information. We appreciate the time that was spent on this experiments and putting all the data together a lot.

    In order to proceed in this case I like to ask you to submit an Abreview for Anti-ISWI (acetyl K753) antibody (ab10748) an our product pagehttps://www.abcam.com/ISWI-acetyl-K753-antibody-ab10748-reviews.html. To do so just follow the link on this page that says "Submit an Abreview".

    Please include all the provided data in this Abreview and our Fast Track Team will contact you as soon as possible and issue you your reward points.

    Thank you very much for participating in our Fast Track program.

    I hope this information was helpful for you. Please do not hesitate to contact me again if you need further help and advice.

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    Answer

    Thank you for your email. I am pleased to hear your customer has been working with the Fast-track antibody ab10748, directed against ISWI (acetyl K753).

    The customer would be eligible to receive Abpoints in return for their efforts which can be converted to credit against future Abcam orders, or Amazon vouchers. In order to claim these Abpoints the customer will need to submit an Abreview with their results.

    This can be done by following the link:

    https://www.abcam.com/index.html?pageconfig=your_reviews&intAbID=10748&strAccessMode=public_create

    I hope this information has been of help. If I can be of any further assistance, please do not hesitate to let me know.

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    Answer

    Thank you for your answer.

    Have you had the opportunity to test the antibody in Western blot as well? Indeed, as you have a positive control, I can recommend strongly to test the antibody in Western blot to see whether the antibody has retained its activity. Additionally,I would recommend also to test thereversed cross-linked ChIP sample in Western blot to see, whether the antibody pulled down the target protein.

    Thank you for sending also the ChIP protocol.It seems you made all the necessary controls andthere are only two points which I could recommend to try to optimize:

    1.) Although the fragment size after DNA fragmentation seems ok, I would recommend to reduce the crosslinking. Indeed, fragment sizes of 1kb might be easier to work with and having a lower degree of crosslinking might help the antibody to bind better the target protein. In our lab, we routinely fix (Hela cells) in 0.75% PFA for 10 minutes and add Glycin afterwards to stop the reaction.

    2.) We would expect the polyclonal antibody to work with 4ul in 300ul. However I can recommend to test either even more antibody or a more concentrated sample. For monoclonal antibodies, we sometimes even test 10ug of antibody for 25ug of chromatin preparation.

    I hope this information is helpful. Please let me know, whether the suggestions helped to improve the results. I look forward to hear back from you.

    Read More

    Answer

    Thank you for contacting us. I am sorry to hear that the results have not been as expected so far.

    Unfortunately, we are not able to test this antibody hereas it is difficult to get the positive controls. This is also why this antibody is still in the "fasttrack" status.

    I would like to recommend to you to test firstly this antibody in Western blotwith a positive control lysate.This would allow to assess whether there is any acetyl K753 ISWI in the sample andto doublecheck whether the antibody itself hasnot been damaged and is still active. As positive control you could use as documented on thedatasheet (1) Recombinant ISWI acetylated with hGCN5 or (2) SL-2 cells

    We do not have a new stock in lot of this antibody unfortunately. If you would like, I can also propose to you tosend us the protocol information for the ChIP experiment andwe couldsee whether there is any suggestion we could make to improve the protocol for this antibody. I have attached the questionnaire to this email, which would help to put the ChIP information together.

    I hope this makes sense. Please let me know if you have any questions. I look forward to hear back from you.

    Read More

    Answer

    Thank you for your enquiry and your interest.

    As the datasheet indicates this antibody recognizes Drosophila melanogaster ISWI (acetyl K753). However, it is not a fully characterized antibody, it is a Fast track antibody. It has been affinity purified and shows high titre values against the immunizing peptide by ELISA.

    Could I advise you to read the Terms and the Conditions on the product datasheet (information in the yellow box):

    Our customers have told us that they want cutting-edge products released quickly so that their research is not impeded. Abcam is therefore pleased to be able to offer researchers access to products that are still in development.

    Fast-Track antibodies constitute a diverse group of products that have undergone variable degrees of characterization. They have all been affinity purified and show high titre values against the immunizing peptide (by ELISA).

    As a general rule, Fast-Track antibodies fall into two main categories:

    Cutting-edge products: these are released quickly to meet customer needs and requirements; the products are still undergoing further tests and characterization steps
    Products which are more complex to characterize due to low levels/lack of expression in easily available lysates or problems experienced in searching for or developing the correct lysate/cell line





    For these reasons, we release Fast-Track antibodies at a reduced price under the following terms:

    As these antibodies have not been fully characterized at Abcam, we cannot guarantee that the antibody will work in any application other than ELISA against the immunizing peptide and they are therefore not covered by the full Abpromise guarantee
    Our experienced Scientific Support Team is happy to offer protocol tips and advice to help improve the results obtained
    Whenever possible, Abcam will also make available data provided by customers that have bought a Fast-Track antibody, the information provided should be used as a guideline only





    Feedback reward
    We would greatly appreciate your feedback on these antibodies, whether positive or negative. We will award 950 bonus Abpoints to the first researcher who sends us positive feedback (including an image and details of materials and methods). Conclusive negative feedback that leads us to withdraw the antibody will also be rewarded with 950 bonus Abpoints. Other useful positive and negative feedback will be rewarded with the standard Abpoints for submitting an Abreview (please refer to Abreviews terms and conditions for more detailed information). All awards are subject to approval by Abcam scientists and provision of suitable data. Please submit feedback to mailto:fast-track@abcam.com



    If you need any further assistance in the future, please do not hesitate to contact me.

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    Answer

    Thank you for your enquiry. I am pleased that you have expressed an interest in this product. This is an in house product and the images are derived from a collaborator. We do not as yet possess any specific published references relating to this product. If you require any further information then please get back in touch with me and I will in turn speak with the business development manager who "owns" this project. I look forward to hearing from you soon.

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    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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