Overview

  • Product name
    Anti-RFP antibody (Biotin)
    See all RFP primary antibodies
  • Description
    Rabbit polyclonal to RFP (Biotin)
  • Host species
    Rabbit
  • Conjugation
    Biotin
  • Specificity
    ab34771 recognises RFP. The antibody is predicted to react with all RFP variants from Discosoma (including DsRed, TdTomato, mOrange etc.) based on sequence homology and polyclonality of the antibody. However, such crossreactivity has not been QC tested.
  • Tested applications
    Suitable for: ELISA, WB, ICC, IHC-P, IHC-Fr, IHC-FrFlmore details
  • Immunogen

    Red Fluorescent Protein (RFP) fusion protein corresponding to the full length amino acid sequence (234aa) derived from the mushroom polyp coral Discosoma.

  • General notes

    Biotin/Protein Ratio: 10-20 BAC molecules per IgG molecule Biotinamidocaproate N-Hydroxysuccinimide Ester (BAC)

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 1% BSA, 0.42% Potassium phosphate, 0.87% Sodium chloride
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Purification notes
    This product was prepared from monospecific antiserum by immunoaffinity chromatography using Red Fluorescent Protein (Discosoma) coupled to agarose beads followed by solid phase adsorption(s) to remove any unwanted reactivities. No reaction was observed against Human, Mouse or Rat serum proteins. ELISA was used to confirm specificity at less than 0.1% of target signal.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab34771 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 1/10000 - 1/80000.
WB 1/2000 - 1/10000. Detects a band of approximately 27 kDa.
ICC 1/1600.
IHC-P 1/1000 - 1/5000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Endogenous tissue biotin should be considered as a source of false-positive staining when immunohistochemical or histochemical techniques which use avidin or streptavidin reagents or anti-biotin antibodies as components of the detection system, are applied to tissue sections. We recommned blocking the endogenous biotins.

IHC-Fr 1/400.
IHC-FrFl Use at an assay dependent concentration. PubMed: 24051358

Target

  • Relevance
    Fluorescent proteins have become a useful and ubiquitous tool for making chimeric proteins, where they function as a fluorescent protein tag. Typically they tolerate N- and C-terminal fusion to a broad variety of proteins. They have been expressed in most known cell types and are used as a noninvasive fluorescent marker in living cells and organisms. They enable a wide range of applications where they have functioned as a cell lineage tracer, reporter of gene expression, or as a measure of protein-protein interactions.
  • Alternative names
    • DsRed antibody
    • GFP like chromoprotein antibody
    • red fluorescent protein antibody
    • RFP antibody

Images

  • All lanes : Anti-RFP antibody (Biotin) (ab34771) at 1/2500 dilution

    Lane 1 : 10 µg of HeLa cell
    extract containing GFP
    recombinant protein
    Lane 2 : 10 µg
    of a HeLa lysate containing RFP recombinant protein.


    Secondary
    All lanes : IRDye™800 conjugated Goat anti-Rabbit IgG at 1/5000 dilution

    Observed band size: 27 kDa
    why is the actual band size different from the predicted?



    IRDye™800 fluorescence image was captured using the Odyssey® Infrared Imaging System developed by LI-COR.
  • ab34771 staining mouse subcutaneous tissue that had been injected with a mixture of cells including mouse embryonic stem cells previously transduced with a retrovirus that expresses RFP.

    The sample was fixed with paraformaldehyde and a heat mediated antigen retreival step using Sodium citrate 10 mM (pH 6.0) was performed.  1.5% serum was used as the blocking agent for 30 minutes at 20°C.  The primary antibody was diluted 1/400 in 1xPBS with 1.5% normal serum and incubated for 1 hour at 20°C. A Biotinylated goat anti-rabbit IgG (H+L) antibody was used as the secondary.

    See Abreview

  • ab34771 staining RFP in transfected Mouse N2a cells by ICC (Immunocytochemistry). Cells were fixed with paraformaldehyde, permeabilized with Triton X-100 and blocked with 5% serum for 2 hours at 25°C. Samples were incubated with primary antibody (1/100 in PBS + 2% BSA) for 12 hours at 4°C. An Alexa Fluor®546-Streptavidin -conjugated anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

    See Abreview

  • Anti-RFP antibody (Biotin) (ab34771) at 1/1000 dilution
    Secondary
    Peroxidase streptavidin secondary antibody at 1/40000 dilution
  • ab34771 staining mouse epidermis that had been damaged and injected with a mixture of cells inlcuding mouse embryonic stem cells previously transduced with a retrovirus that expresses RFP.  The color is developed with DAB.

    The sample was acetone fixed and incubated with ab34771 diluted 1/400 for 1 hour at 20°C. A Biotinylated goat anti-rabbit IgG (H+L) antibody was used as the secondary.

    See Abreview

  • ICC image of ab34771 staining murine embryonic stem cells, transduced with RFP gene, cultured over mouse embryonic fibroblast.  The cells were PFA fixed and blocked with 1.5% serum for 45 minutes at 20°C.  The primary antibody was diluted 1/1600 and incubated for 1 hour and 15 minutes at 20°C. A biotinylated goat anti-rabbit IgG (H+L) was used as the secondary.

    See Abreview

References

This product has been referenced in:
  • Sun J & Zheng H Efficient ER Fusion Requires a Dimerization and a C-Terminal Tail Mediated Membrane Anchoring of RHD3. Plant Physiol 176:406-417 (2018). WB . Read more (PubMed: 29138354) »
  • Hu X  et al. Autonomous Magnetic Microrobots by Navigating Gates for Multiple Biomolecules Delivery. Small 14:e1800504 (2018). Read more (PubMed: 29740954) »
See all 44 Publications for this product

Customer reviews and Q&As

1-10 of 30 Abreviews or Q&A

Answer

Thank you for contacting us.

The buffer we have used is Sodium citrate buffer 6.0 pH. The heat mediated antigen retrieval method should be optimized by the end user.

We recommend incubating the sections for 5,10, or 15 minutes in citrate buffer in 700 watt Microwave. In case of pressure cooker at high pressure 2 minutes incubation would be enough.

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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Question
Answer


The unconjugated antibody is ab124754.
https://www.abcam.com/rfp-antibody-ab124754.html


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Answer

Thank you for your reply and the detailed information provided. This will be very useful to investigate this case.

It seems that regardless of the blocking conditions used, with the antibody ab62341 you are seeing very high background. This is quite blotchy, leading me to think that there are particles in one of the buffers which is sticking non-specifically to the membrane. It could be that the exact origin of this could be ascertained by testing all the different buffers, but as I suspect you have been using the same blocking and wash buffers for the ab34771 (where this background was not observed) and ab62341 I think further investigation would not be warranted.

It may be that this blotchyness could be removed by filtering your solutions prior to their use. However, I can understand that you have already put a lot of effort into optimising this antibody. I would therefore be happy to provide a replacement vial of (ab62341), or if you would prefer, a different antibody altogether. I would also be able to arrange a credit note or refund if this is preferable to you.

In order to arrange this, could you please provide me with the order number of ab62341 (or if you do not have this information, the delivery address)?

I look forward to receiving your reply.

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Answer

Thank you for your email.

You are right; I meant the 04/04/13 of course. I can reassure you that the code is valid until 04/04/13.

I wish you good luck with your experiment and look forward to hearing about your experience with this antibody in Flow Cytometry.

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Answer

DISCOUNT CODE: ABTRIAL-xxxxx
Expiration date: 04/04/12
Value: €410.00

I am very pleased to hear you would like to accept our offer and test ab34771 in Flow Cytometry. This code will give you €410.00 off your next order before the expiration date. To redeem this offer, please submit an Abreview for Flow cytometry and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.
For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.


It is correct, that you do not have to mention the code with your order of ab34771. The code will be activated by submitting the Abreview and needs to be mentioned with the next order.

Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/abtrial.

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Answer

Thank you for your answer.

I would be pleased to offer you the AbTrial for the ab34771 to use this antibody in FlowCytometry. If you decide to order this antibody please let me now in advance so that I can provide you with the code.

You will receive the whole value of the antibody you are testing to order a new product. That means, if you decide to test the ab34771 you will receive a code with a value of €410.00. The code will be activated after you submit the Abreview.

Please let me know about your decision. I look forward hearing from you.

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Answer

Thank you for contacting us.

In this case I would recommend to use the Anti-RFP antibody (ab62341). If it is suitable for your experiment you could use a secondary antibody to detect the Anti RFP antibody.

If the conjugate needs to be on the primary antibody I would recommend to have a look at our conjugation kits https://www.abcam.com/index.html?pageconfig=resource&rid=13148.

Because the Anti-RFP antibody (ab62341) has not been tested for flow cytometry I would am pleased to offer you the AbTrial for the use of this product in flow cytometry. By participating in our AbTrial program you can use our products in an untested application or species without financial risk.

Simply follow these easy steps below to apply for our AbTrial Program:

1. Reply to this email, letting me know you are interested in testing this product.

2. I will email you an inactive personal discount code for the value of the product.

3. Purchase and test the product at the regular price.

4. Submit your results, including your discount code in the additional notes section of your Abreview.

5. Once the Abreview is submitted, the discount code will become active.

6. Apply your discount code on your next order to receive that value off.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/abtrial.

I hope I could be of help and I look forward hearing from you about your decision regarding the AbTrial offer.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Answer

Thank you for your reply.

We attempt to list all the information that we have about any of our products on the datasheet provided the information is not commercially senstive. Currently we are not aware of ab34771 being tested in transgenic DsR mice. Ab34771 is predicted to react with all RFP variants (including DsRed, TdTomato, mOrange etc.) based on sequence homology and polyclonality of the antibody. However, such crossreactivity has not been QC tested.

The product has been tested against GFP as shown in the Western blot image on the datasheet. In this experimentHeLa cell extract containing GFP recombinant protein andHeLa lysate containing RFP recombinant protein where compared. There was no reaction against GFP.

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 contacting us with your question.

Ab34771 is conjugated to biotin so that a biotin-streptavidinsystem can be used in the detection of the antibody, which will amplify the signal. To use this antibody in immunofluorescent staining in IHC, you would usestreptavidin conjugatedto a fluorophore instead of the secondary antibody step. We have several streptavidinconjugatesin our catalog that can be used with Ab34771, such as ab13622 which is conjugated to FITC-

https://www.abcam.com/Streptavidin-protein-FITC-ab136222.html

If you prefer an unconjugated RFP antibody to use with a secondary conjugated antibody, we also have Ab62341 which has been tested with frozen and paraffin-embedded tissue in IHC-

https://www.abcam.com/RFP-antibody-ab62341.html

Ab62341 has also been referenced in 10 Abreviews and 11 publications, so I think this will be a good choice for your staining.

I hope that this information will be useful, but please let me know if you have further questions and I'll be happy to help.

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Question
Answer

Thank you for your patience.



I have enquired with the product originator but unfortunately, the actual sequence information isproprietary and cannot be released. They did however mention that the sequence wasderived from thereference links below:


http://www.ncbi.nlm.nih.gov/protein/55976617?report=genbank&log$=prottop&blast_rank=2&RID=56D9X48A01N

http://www.uniprot.org/uniprot/Q9U6Y8

I hope this information will still be helpful to you.

All of our products are guaranteed to work in species and application as stated on the datasheet. Should you not get expected results, then please inform us within the guarantee period of 6 month (Abpromise). We will help troubleshoot the experiment and if the product is indeed faulty, we will be more than happy to offer a free replacement or refund.

I hope our Abpromise guarantee will give you the assurance you need to use this product in your research.

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1-10 of 30 Abreviews or Q&A

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

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