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  1. Link

    fitc-rabbit-igg-isotype-control-ab37406.pdf

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Isotype/Loading Controls Isotype Controls Rabbit
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FITC-Rabbit IgG  - Isotype Control (ab37406)

  • Datasheet
  • SDS
Q&A (15)References (5)

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ELISA - FITC-Rabbit IgG  - Isotype Control (ab37406)

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    Overview

    • Product name

      FITC-Rabbit IgG  - Isotype Control
      See all Rabbit isotype controls
    • Conjugation

      FITC. Ex: 493nm, Em: 528nm
    • Specificity

      This antibody has been selected to be an isotype control as it has no known specificty.
    • Tested applications

      Suitable for: ELISA, Flow Cytmore details
    • General notes

      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 this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.

      If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As

    Properties

    • Form

      Liquid
    • Storage instructions

      Shipped at 4°C. Store at +4°C.
    • Storage buffer

      pH: 7.40
      Preservative: 0.1% Sodium azide
      Constituent: PBS
    • Concentration information loading...
    • Purity

      IgG fraction
    • Clonality

      Polyclonal
    • Isotype

      IgG
    • Research areas

      • Isotype/Loading Controls
      • Isotype Controls
      • Rabbit
    • Alternative names

      • rabbit isotype control

    Associated products

    • Compatible Secondaries

      • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
      • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)

    Applications

    The Abpromise guarantee

    Our Abpromise guarantee covers the use of ab37406 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
    ELISA
    Use at an assay dependent dilution.
    Flow Cyt
    Use 10µl for 106 cells.
    Notes
    ELISA
    Use at an assay dependent dilution.
    Flow Cyt
    Use 10µl for 106 cells.

    Images

    • ELISA - FITC-Rabbit IgG  - Isotype Control (ab37406)
      ELISA - FITC-Rabbit IgG  - Isotype Control (ab37406)

      FLISA plate was coated with Goat Anti-Rabbit IgG-UNLB and Goat Anti-Rabbit IgM-UNLB.  Serially diluted Rabbit IgG-FITC ab37406 was captured and fluorescence intensity was quantified.

    Protocols

    • Flow cytometry protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (5)

    Publishing research using ab37406? Please let us know so that we can cite the reference in this datasheet.

    ab37406 has been referenced in 5 publications.

    • Kitao A  et al. Band 3 ectopic expression in colorectal cancer induces an increase in erythrocyte membrane-bound IgG and may cause immune-related anemia. Int J Hematol 111:657-666 (2020). PubMed: 31997080
    • Landa-Solís C  et al. Behavior of multipotent stem cells isolated in mobilized peripheral blood from sheep after culture with human chondrogenic medium. Tissue Cell 52:116-123 (2018). ICC/IF ; Sheep . PubMed: 29857820
    • Daringer NM  et al. Modular extracellular sensor architecture for engineering mammalian cell-based devices. ACS Synth Biol 3:892-902 (2014). ICC/IF . PubMed: 24611683
    • Gerlach JC  et al. Perivascular Mesenchymal Progenitors in Human Fetal and Adult Liver. Stem Cells Dev : (2012). PubMed: 22931482
    • Mishra PK  et al. Exercise mitigates homocysteine - ß2-adrenergic receptor interactions to ameliorate contractile dysfunction in diabetes. Int J Physiol Pathophysiol Pharmacol 3:97-106 (2011). PubMed: 21760968

    Customer reviews and Q&As

    Q&A
    Submit a question

    1-10 of 15 Q&A

    Question

    Name: Rachel Dudek

     

    Inquiry: Hello, I am wondering if you could provide me with the F/P ratio for antibody ab37406, a Rabbit IgG FITC - Isotype Control, so I can compare this to the F/P ratio of my specific antibody? The lot number that I have is GR125056-2. Thank you for your help.

    Read More

    Abcam community

    Verified customer

    Asked on Nov 22 2013

    Answer

    Thank you very much for contacting us with your question, and for your patience.
    I have contacted the lab scientists, and the F:P ratio is between 3 and 6 for this antibody.
    I hope this information is useful, but please let me know if you have further questions or if there is anything else that we can do for you.

    Read More

    Caitlin Valued Customer

    Abcam Scientific Support

    Answered on Nov 22 2013

    Question

    I have received today the product "Rabbit IgG FITC-Isotype Control (ab37406)" and I note that the storage instructions are changed.

    In fact, in the data sheet of the previous aliquots, the storage instructions reported: SHIPPED AT +4°C. UPON DELIVERY ALIQUOT AND STORE AT -20°C or -80°C. AVOID REPEATED FREEZE/THAW CYCLES.

    Now is reported only: STORE AT +4°C.

    How I have to store the antibody? Thank you in advance.

    Read More

    Abcam community

    Verified customer

    Asked on Jul 19 2013

    Answer

    We have done stability studies by storing conjugated antibodies at different temperature. The study shows FITC conjugated products are more stable at 4C rather than -20C so in response to this we changed the datasheets of all conjugated antibody we have in catalogue.
    FITC conjugate will be stable for 2 years at 4C.
    Please note the Abpromise guarantee period is 1 year from date of purchase.

    Read More

    Padamjeet Singh

    Abcam Scientific Support

    Answered on Jul 19 2013

    Question

    Inquiry: I have purchased the product with details below. Can you please provide me the concentration and the volume of the the product? What is your recommended concentration to use for flow cytometry using frozen peripheral blood mononuclear cell (PBMC)? Rb IgG FITC Isotype control (100 test) (IgG Fraction) Ab37406 Lot GR 59579-1 Regards,

    Read More

    Abcam community

    Verified customer

    Asked on Oct 04 2012

    Answer

    Thank you for contacting us and your interest in our products.

    The Rabbit IgG FITC - Isotype Control (ab37406) of the lot number GR59579-1 is provided at a concentration of 0.1mg/mL and is supplied as 1 mL.

    As the antibody is intended to be used as an isotype control we would advise using the antibody with the same protocol you intend to use with the rabbit IgG antibody you are using to detect a target in your experiment.

    I hope this information has been of help. If you have any further questions, please do not hesitate to contact us again.

    Read More

    Abcam Scientific Support

    Answered on Oct 04 2012

    Question

    Hi,

    I have purchased ab37406 lot number GR77642. I need to use this alongside other antibodies at a known concentration.

    Please could you tell me the concentration of this antibody.

    Regards,

    Read More

    Abcam community

    Verified customer

    Asked on Sep 06 2012

    Answer

    Thank you for your inquiry.
    I am happy to confirm that ab37406 (lot GR77642) has a concentration of 0.1mg/ml.
    I hope this information is helpful and wish you good luck with your research.

    Read More

    Abcam Scientific Support

    Answered on Sep 06 2012

    Question

    For the below antibodies I am looking for both isotype control and positive controls. It would be great if you could give the information for both in case you have them.
    ab36204 --> Rabbit IgG
    ab48614 --> Rabbit IgG
    ab66219 --> Rabbit IgG
    ab83508 --> Mouse IgG1
    ab102998 --> Rabbit IgG
    In the list I was asking for isotype controls, but still the positive controls would be interesting to know, too.
    All the best,

    Read More

    Abcam community

    Verified customer

    Asked on Apr 03 2012

    Answer

    Thank you for your clarification.

    I can confirm the concentration of ab66219 (Batch: GR9812) of this ab is 1 mg/ml. The other antibody ab36204 (Lot: GR42382) is not affinity purified but whole antiserum so the IgG is not quantified. However, I can provide a range of the IgG: 10-15 mg/ml.

    Isotype controls:



    can be searched at this site: https://www.abcam.com/index.html?c=718

    ab36204, ab48614, ab66219, ab102998 --> Rabbit IgG - ab37406, ab125938, ab128142

    ab83508 --> Mouse IgG1 - ab91353, ab27479, ab81032



    Positive control:



    ab36204 - CRMP4 - It is mainly expressed in heart and skeletal muscle and also strongly expressed in fetal brain and spinal cord (human, mouse or rat),

    ab48614 --Ceruloplasmin - It is expressed by the liver and secreted in plasma,

    ab66219 -- Oprl1 - Brain (cortex),

    ab83508 -- NG2 - It is detected only in malignant melanoma cells, SK-MEL-28 cells,

    ab102998 --B3GNT5- only tested in WB - Widely expressed. It is highly expressed in lung, colon, placenta, testis, pituitary gland and cerebellum, MDA-MB435 cell line

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

    Read More

    Abcam Scientific Support

    Answered on Apr 03 2012

    Question


    Thank you for the reply.
    In answer to your questions:
    1. Unfortunately I have no data supporting the expression levels of
    the CDT1 in this particular cell line so it's definitely worthwhile to
    stain HeLa cells and see whether the weak fluorescent signal arises
    due to the low concentration of CDT1 within my cells.
    2. The cell viability is checked by means of the trypan blue exclusion
    method, about 1-2% of cells are dead as they don't efflux effectively
    the dye, therefore I've confirmed in every sample prep that the cells
    are viable. Also, the cells are seeded at a concentration of 40,000
    cells/ml (concentration that has shown to give place to a log growth).
    The ration cells per area is: 12,500 cells/cm2 (which has demonstrated
    by previous studies that enable the log growth of the cells).
    3. Unfortunately the secondary antibody hasn't been used with another
    primary antibody and the primary antibody hasn't been detected by
    another secondary antibody -this is the only system I currently have,
    but I do see the point of checking the actual effectiveness of both so
    I'll try to get some aliquotes from my collegues and see what sort of
    staining profile I get-.
    4. I'll run the experiment you suggested next week (as soon as I get
    some HeLa cells). And I'll also grow the cell line of interest
    (Caki-2) and perform the staining on the glass they've growth on so we
    can conclusively know whether the trypsin is having some detrimental
    effect on the staining.
    At the moment I'm staining my last batch of samples (fixed with 50%
    methanol and 50% acetone). The samples are going to be incubated with
    the primary antibody overnight at 4C and hopefully the specific signal
    from the positive control will be better.
    Best regards,
    PS. As I'm not an expert on the art of performing ICC nor in biology,
    I've been reading about those things that might potentialy affect the
    staining and I've noticed that the majority of the antibodies are
    aliquoted and stored either at -20 or -80 C. When I first got my
    antibodies, I followed the storage instructions for all of them;
    therefore I aliquoted both: the primary antibody (ab83174) and the
    Rabbit Isotype Ctrl (ab37406) and then I stored them at -20 C. I also
    followed the storage instructions for the secondary antibody (ab60314)
    shown in both the datasheet that the antibody came with and the Abcam
    website for this particular antibody so I've kept it at 4C. I wonder
    if perhaps there is a mistake on the actual storage instructions and
    therefore this antibody has gone bad as it's been kept @ 4C for about
    4 months by now?
    Sorry for the silly question but at this point I'm trying to track any
    potential failure in the whole system as I have not got a successful
    staining profile yet.
    Thank you in advace.

    Read More

    Abcam community

    Verified customer

    Asked on Mar 29 2012

    Answer

    I think its good that you are checking the cell viability and using HeLa may provide more information as to if it is the cell strain or the antibodies that may be contributing to the problems. I agree that checking both the primary and secondary antibody is excessive but if you have access to an alternative secondary antibody it may be worthwhile trying. The activity of the secondary antibody should not have been compromised as you are storing it as we have recommended (and I have checked that these guidelines are correct). The antibody should also be stored in the dark, as should ab37406. Ab37406 and ab83174 should be aliquotted (we usually recommend no lower than 10 uL) and stored at -20 or -80. After thawing an aliquot this should not be refrozen but can be storedat 4degreesfor a few weeks if some is left over after your experiment.


    Let me know how you get on with your experiments. I'll be crossing my fingers for you.

    Read More

    Abcam Scientific Support

    Answered on Mar 29 2012

    Question

    Hi xxxx,

    Firstly I'd like to thank you for all the suggestions you've made so far, it's been a very nice Abcam experience and the better technical support I've ever got. So, thank you again.

    You are right, now we now that there is definitely occuring non-specific binding of both antibodies.

    Cytospinning or growing the cells onto the substrate.
    The renal cell line I'm working with is called Caki-2 and it is adherent. Even though the cells DO grow onto the substrate (CaF2 slides without any sort of coating) they tend to grow very close to each other -even after just 24 hours of being seeded at a low cell density - making clumps of cells that are not useful for me any more as I'm planning to collect spectroscopic data from isolated "single-cells" before actually performing any ICC on them (which takes about 2 days) >> that's also the main reason for which I fix the cells and leave them dry (water is a strong absorber of IR radiation).

    The special substrate that I need for collecting the spectra isn't particularly "friendly" with the cells, therefore some toxicity might arise from it. Putting a coating of either L-lysine or some other thing onto the surface would mask further slight spectral variations due (for example) cytotoxicity of anticancer drugs or cell-cycle (that I'm planning to detect). As the nature of the substrate isn't completely suitable for growing the cells onto it I need therefore to cytospin them down onto the surface of something IR inactive (in this case CaF2 windows) that enable me to collect useful spectral information without actually interfering with the inherent chemistry of my treated cells.


    Fixative
    The reason for which I chose to use PFA as cell fixative instead methanol fixation relies on the fact that by using the later one some lipids from the membrane may be removed (and by means of spectroscopy it will be not ideal to have a cell without a complete membrane).
    As I'm planning to collect IR data from my samples and then ICC stain them I can not keep them in a hydrated form and perform ICC immediately.

    However, what I'll do following your suggestion will be to run a parallel ICC test on the next batch of samples with the same concentration of antibodies and see whether the fixation process is a contributing factor for giving place to the non-specific binding. If the results are definitely better with methanol then I will modify the overall protocol in order to get the most out of the ICC staining (which is the important bit at the moment!!). =)

    I will also fix the cells after they have been cytospun as you suggested.
    You were right, the antibodies have always been diluted in 1% BSAin PBST.

    I'll go back to you once I get the next experimental results -hopefully they will be brilliant!-

    Best wishes,

    xxxxxxx

    Read More

    Abcam community

    Verified customer

    Asked on Mar 14 2012

    Answer

    Thank you for providing such a full explanation of what you are trying to achieve. I can now understand exactly why you have chosen the technique that you have.

    I think the experiments you are planning to do next will be very useful in determining how best to proceed with the staining and more fully understandingwhere the non-specificity iscoming from.I too hope they will be brilliant! I don't knowif you areplanning on it yet but I would suggest keeping at least oneof your test withoutair dryingyourcells(i.e keeping them hydrated throughout), just to seeif thisimproves the non-specificity seen with the secondary and isotype control antibodies. If it does, it would make youractual experimentdifficult to measure theIR butI think it is most important at the moment toget the specific staining. Once this has been achieved, the optimisationof how toperform the actual experiments can be done.

    I wish you all the best with the new experiments and look forward to hearing how you get on.

    Read More

    Abcam Scientific Support

    Answered on Mar 14 2012

    Question

    Dear xxxx,

    I contacted you a few weeks ago as I'm having having some problems for stainng my samples with the following ICC system:
    Primary antibody: Anti-CDT1 antibody (ab83174)
    Secondary Antibody: Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Chromeo™ 488) (ab60314)
    Control Isotype: Rabbit IgG FITC - Isotype Control (ab37406)
    I performed the ICC staining of my samples last week by following all the useful suggestions you made in order to optimize the protocol and unfortunately I still get signal coming from the controls.
    Please find attached to this email a .ppt file that describes the experiments I conducted and the images collected after staining my samples.
    I wonder if you could provide some advice in terms of what to do under these circumstances.
    Any information will be much appreciated.

    The modifications of the protocol were the following:

    Formalin fixation: Yes. Time: 15 minutes (rather than one hour).
    Blocking time: 45 minutes with 10% Goat serum in 1%BSA in PBS (rather than 30 minutes).
    Incubation time for each antibody: 1 hour at room temperature (rather than overnight at 4C)
    Dilution of the primary antibody: 5 ug/ml (as recommended).
    Dilution of the secondary antibody: 1:1500 and 1:2000 (1.4 and 1 ug/mL respectively rather than 4ug/mL).

    Also, my supervisor suggested to hydrate the samples before doing the actual ICC staining by incubating them with PBS for 30 minutes, I wonder if perhaps this was not a good idea and may be a contributing factor for giving place to the misbinding?

    Best wishes,

    Read More

    Abcam community

    Verified customer

    Asked on Mar 13 2012

    Answer

    Thank you for getting back to me with your new results. It is good that we can now say conclusively that there is non-specific binding of both the secondary antibody and of the isotype control antibody.

    Following the protocol changes, I do not exactly know why this non-specific nuclear staining is persisting but I have a few suggestions to add that may be beneficial.

    I am not an expert in using cytospin by any means so I have been having a read about it. It seems that it is generally recommended to first spin your suspension, then fix the cells on the slides. The following reference may be of help to you:

    http://www.ihcworld.com/_protocols/histology/cytospin.htm

    http://sciencepark.mdanderson.org/labs/tang/cytospin4web.html

    It may also be beneficial to fix immediately, then stain immediately. I would not allow your cellsto dry out at any stage.The only Abreview we have of ab83174 having been used in ICC used PFA fixation and some non-specific staining of the cytoplasm was also seen (which seems to be what you are seeing). We have charaterised this target using methanol fixation (refer toab83174 datasheet). It maybe worthwhile to try this in order to improve the staining.

    Is there a reason why you are using cytospin? If your cells are adherent you could just grow the cells on glass slides directly as is described in the following protocol:

    https://www.abcam.com/index.html?pageconfig=resource&rid=11417

    I have a further question. What diluent are you using for your antibodies? I would suggest still usingthe same asyouwere (1% BSAin PBST).Are you performingany washing steps? Following each incubation I would wash theslide with PBS 3 times for 5 minutes.

    If you have any questions in regards to my suggestions please do let me know.

    Read More

    Abcam Scientific Support

    Answered on Mar 13 2012

    Question

    Dear xxxx,

    The information provided is much appreciated, thank you very much for all the suggestions made. I think that definitely there are opportunities for improving my protocol.
    In answer to your question, the preparation of the diluted isotype control was performed as follows:

    Initial concentration: 0.1mg/mL
    Desired concentration: 5 micrgrams/mL

    In order to get 0.5 ml of the desired concentration 25 microL of the original Isotype Control were diluted in 475 microL of 1% of BSA in PBST.

    Thank you again for the technical support. I'll start the next set of experiments as soon as possible and I'll get back to you with the new results!

    Regards,

    Read More

    Abcam community

    Verified customer

    Asked on Feb 16 2012

    Answer

    Thank you for that last bit of information.


    I just wanted to check that the isotype control wasn't being used in a higher concentration than the primary antibody, but everything is fine.


    I look forward to hearing how you get on.

    Read More

    Abcam Scientific Support

    Answered on Feb 16 2012

    Question

    Dear xxxx,

    Please find attached to this email the images corresponding to both, the positve staining controls and the actual controls for discarding misbinding of the antibodies.
    Even thouh I'm showing just one set of images the general trend is the same for the replicates.

    You can find below this email the questionnaire.
    At the bottom of it I detailed what I'm planning to do next, however any suggestion you may have in order to improve the protocol will be most welcome!

    Thank you in advance,

    Looking forward to hearing from you.

    Read More

    Abcam community

    Verified customer

    Asked on Feb 16 2012

    Answer

    Sorry for the delay in getting back to you and thank you for all that information. It makes it much easier to understand what is going on and what has been tried previously.

    I think the next step you are suggestingare good. The blocking buffer and antibody diluent you are using are both what were used in performing inhouse testing with this antibody. Additionally, performing the following controls:

    1. isotype control only

    2. secondary antibody only (no-primary control)

    3. primary antibody+secondary antibody

    you will see more accurately what element is contributing to the non-specific signal.

    There are a few things I would add though.

    1. for fixation with formalin we would normaly say 10-15 minutes would be enough. The danger in prolonging the fixation is that the epitope that the antibody recognises can be masked, requiring antigen retrieval to be employed. It can also lead to increased non-specific binding. I would therefore suggest reducing the fixation time.

    2. the recommended dilution for the secondary antibody you are using is 1/1000 to 1/2000. This is equivalent to 1-2 µg/mL. I would therefore reduce the concentration used from 4 µg/mL to 1 µg/mL, which will hopefully help in reducing the non-specific signals observed.

    Additionally, may I ask how thedilutionof the isotype control was performed?

    The isotype that you are using I would not specifically recommend as a control for the experiment you are performing. As the antibody is already FITC conjugated, if it does bind non-specifically and the secondary also binds, you will get an amplification of the signal over and above what the actual primary antibody non-specificity would yield. I would either suggest using a primary antibody that is directly FITC conjugated, or an isotype control such as ab27478.

    Ihope the suggestions I have made improve the results seen so far. I look forward to hearing how you get on.

    Read More

    Abcam Scientific Support

    Answered on Feb 16 2012

    1-10 of 15 Q&A

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