Overview

  • Product name
  • Description
    Rabbit polyclonal to RFP
  • Host species
    Rabbit
  • Specificity
    ab28664 detects RFP tagged proteins. This antibody is specific for the RFP tag. It shows very low reactivity to GFP or GFP tagged proteins. ab28664 non-specifically detects a 45 kDa protein from E. coli extracts. This non-specific cross-reactivity is not present in mammalian samples.
  • Tested applications
    Suitable for: WB, ICC/IFmore details
  • Immunogen

    Synthetic peptide:

    VNGHEFEIEGEGEGR

    , corresponding to amino acids 22-36 of RFP from Discosoma sea anemone.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage buffer
    Preservative: 0.05% Sodium azide
    Constituents: PBS, 0.1% BSA
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab28664 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
WB 1/1000. Predicted molecular weight: 26 kDa.

Use at a concentration of 2 µg/ml. Block membrane in 5% milk and incubate primary and secondary antibodies in milk.

ICC/IF 1/50 - 1/200.

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

  • Western blot of recombinant RFP using ab28664.

  • Immunofluorescent analysis of recombinant Red Fluorescent Protein (RFP)-transfected HeLa cells. Cells were transfected with a pCMV RFP C-Myc plasmid and 48 hours post-transfection cells were fixed with formalin and permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature. Cells were blocked with 3% BSA for 15 minutes at room temperature, and then probed with ab28664 at a dilution of 1/50 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight® 488-conjugated goat anti-rabbit IgG secondary antibody at a dilution of 1/400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye. Images were taken at 20X magnification.

References

This product has been referenced in:
  • Lapierre LA  et al. Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition. Mol Biol Cell 28:1088-1100 (2017). Read more (PubMed: 28228550) »
  • Nakagawa N  et al. APC sets the Wnt tone necessary for cerebral cortical progenitor development. Genes Dev 31:1679-1692 (2017). Read more (PubMed: 28916710) »
See all 9 Publications for this product

Customer reviews and Q&As

11-19 of 19 Abreviews or Q&A

Question
Answer

Merci de nous avoir contactés. Malheureusement nous n'avons pas d'anticorps qui ait été développé spécifiquement contre la mCherry. Nous avons par contre un anticorps anti-RFP qui semble bien convenir pour la détection de mCherry : le polyclonal de lapin ab28664, testé en Western Blot, dont l'immunogène présente 100% d'homologie avec la mCherry. Le lien vers la fiche technique de ab28664 est : www.abcam.com/ab28664 . J'espère que ces informations vous seront utiles. N'hésitez pas à nous contacter de nouveau si vous avez d'autres questions.

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Application
Western blot
Sample
Mouse Cell lysate - whole cell (C2C12)
Loading amount
10 µg
Specification
C2C12
Gel Running Conditions
Reduced Denaturing (4-20% gel)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Nov 01 2011

Answer

Thank you for your reply. I have sent a free vial of ab34767 to replace ab28664. I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I have also issued a credit note for ab6722. The credit note may be used in one of the following ways: (1) Redeemed against the original invoice if this hasn't already been paid. (2) Held on the account for use against a future order. (3) A full refund can be offered where no other invoices are outstanding. Please contact your Finance department to confirm how you would like the credit note to be used and ensure it is not redeemed without your knowledge. To specifically receive a refund please ask your Finance department to contact our Finance department at creditcontrol@abcam.com or by telephone using the information at the “Contact Us” link in the top right corner of our website. The credit note ID is for your reference only, please refer to the credit note ID in any correspondence with our accounting department. We will send you the completed credit note by email or postal mail with the actual credit note number which will start with the letters CGB. I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service should you require further expert advice

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Answer

Thank you for your email. I would be happy to send a free vial of ab34767 (to replace ab28664) aswell as credit you for the secondary antibody purchased. Do you have the secondary product code and date of order? I need to find the original order to process the credit. Looking forward to your reply.

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Answer

Thank you for the further information. My colleague Kate is currently out of the office, so I will be managing this case. I am sorry to hear you have been experincing problems with ab28664. I would be happy to send you a replacment of another product, ab34767 may be of interest as it is conjugated with HRP: Click here (or use the following: https://www.abcam.com/index.html?datasheet=34767). Please let me know whether you would like to receive this antibody as a replacement. Reviewing the protocol information, I would be careful about storing the supernatant from the protein extraction at 4C, -20 may be the most appropriate storage temperature to prevent protein degradation. It would also be interesting if there is another method to confirm that the proteins are expressed. Do you have a control protein that can be expressed at the same time and testing with another antibody? This would act as a positive control. I look forward to your reply.

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Answer

Thank you for your enquiry. Yes, ab28664 has been tested for Western blotting and will demonstrate the expression of your DSred fusion protein. But I recommend ab34767 which is an HRP conjugate, removing the need for a secondary detection antibody. It can be used more dilute than ab28664, making it cheaper per blot. It has been tested for ELISA, which will be a more sensitive assay for the purposes of normalization than Western blot densitometry, though Western blot will demonstrate the specificity of the antibody, revealed by a single band for the fusion protein. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for willing to try with 5% BSA. All the information we have on this product is already stated on the datasheet. I am afraid that we do not know of a suitable positive control for this product. I would suggest checking the latest publication on the subject. I am sorry I can't be of more assistance in this occasion but should you need any other information, I will be here to help you.

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Question

BATCH NUMBER 237752 ORDER NUMBER 198485 DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE Media was collected from 293A cells expressing the three chains of laminin 5 (including alpha 3 chain tagged with mCherry). The media was placed on a Ni column and the recombinant protein (containing the beta 3 tagged with His) purified and dialyzed. PRIMARY ANTIBODY Abcam RFP 1:500 in milk incubated at 4 overnight DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED At this point, I don't have anything to use as a positive control. However, on the same blot I probed with other antibodies which recognized the protein itself. ANTIBODY STORAGE CONDITIONS antibody aliquoted and stored at -20 SAMPLE PREPARATION 3X sample buffer (glycerol, SDS, and tris) was added to the sample and then boiled for 3 minutes. AMOUNT OF PROTEIN LOADED 2 micrograms purified protein ELECTROPHORESIS/GEL CONDITIONS 7.5% reducing gel. TRANSFER AND BLOCKING CONDITIONS transferred 2 hours, blocked in 5% milk/PBS for 2 hours SECONDARY ANTIBODY Cell signaling goat anti rabbit 1:500 for two hours. washed 4X over 30 minutes before and after adding secondary antibody in PBS/.05% tween HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No WHAT STEPS HAVE YOU ALTERED? When we first bought the antibody, another lab member tried using it with mixed results. Sometimes there were extraneous bands and other times nothing. This past week I retried it and drew a blank. I have not retried it, but will do so if you can provide some suggestions.

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Answer

Thank you for your enquiry. I am sorry to hear that you are experiencing difficulties with this product ab28664 in western blot. Often it is possible to make suggestions that help resolve problems. We will happily offer technical support but please bear in mind that you 120 days Abpromise guarantee has already expired and therefore, we can not offer you a replacement or refund. If you would still like some technical help, I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem. If the suggestions do not prove to be helpful, would you please be so kind to confirm the following items in order to help me better understand the cause of the problem. In general, most customers who use milk as a blocking agent tend to have problems detecting bands. At Abcam, almost all of our products have been tested with 5% BSA. Sometimes, a different blocking buffer might cause cross reaction between the blocking agent and antibodies, therefore causing weak bands or no bands at all. I would recommend trying 5% BSA, if you have not already done so. Also, cross reaction between the antibodies with the diluent could also cause the lack of signals. We would normally use 1% BSA to dilute the primary and secondary antibodies. You can also use PBST only. Please reduce and denature the sample at 95oC for 10 minutes in buffer containing SDS and mercaptoethanol. This will ensure that the protein is in the correct conformation to run at the correct molecular weight and be detected by the antibody. Can you confirm that the second antibody you used also managed to produce good results with other primary antibodies? In some cases, the problem may be with the secondary antibody not working. Was the washing done 30 min x 4 times? We recommend washing for 15 min x 3 times. This is because over washing will eventually cause you to lose signals. Could the no signal you are experiencing be due to poor transfer of protein to membrane? Did you check the transfer efficiency? This can be simply done with a reversible stain such as Ponceau S. At what volume was the product aliquotted into? Normally, we will aliquot into more than 10ul per vial as it is more stable. If the above suggestions are not helpful, then I am afraid that there is nothing else that I can do to help. I am sorry I can’t be of more assistance in this occasion.

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Question

BATCH NUMBER 218464 ORDER NUMBER 184906 DESCRIPTION OF THE PROBLEM very faint band of monomeric RFP at the expected size of 25kDa. Additional band(s) also in negative control. This faint specific signal plus additional bands is not acceptable for further experiments SAMPLE Crude extract from transient transfected human melanoma cells (MelJuSo) with mRFP-N1 (Invitrogen GFP-N1 vector backbone). Note: mRFP fluorescence (not from immunelabeling with anti-RFP !)was detected with confocal microscope in parralel experiment. PRIMARY ANTIBODY Rabbit polyclonal anti-RFP (Abcam ab28664). Recommended concentration 2ug/ml (1:500 dilution). Applied concentrations: 4ug/ml (1:250 dilution) and 2ug/ul (1:500) Buffers: PBS 0.05%-0.1% Tween-20 (pH 7.2), TBS 0.05% Tween-20 (pH 8.0) Incubation time: 1hr at room temperature, shaking Wash steps: 3 times 10min in corresponding buffer DETECTION METHOD ECL 1, 2 and 5 min exposure POSITIVE AND NEGATIVE CONTROLS USED negative control: extract from untransfected cells no positive control available ANTIBODY STORAGE CONDITIONS Aliquoted and stored at -20 oC SAMPLE PREPARATION Transfected cells were trypsinized after 24hrs, and pelleted in Eppendorf cup. Cells were washed once with PBS. Cells were lysed by addition of 1x SDS PAGE sample buffer (containing SDS, b-mercaptoethanol). Subsequently, the samples were boiled for 5min. AMOUNT OF PROTEIN LOADED 30-50ug ELECTROPHORESIS/GEL CONDITIONS 10% SDS-PA gel...reducing. front of samples was still on gel before transfer. TRANSFER AND BLOCKING CONDITIONS Wet transfer using Biorad system. Transfer buffer: 3.1 gram Tris-base, 11.3 gram glycine, 200ml Methanol in 1L. Nitrocellulose or PVDF membrane. Transfer for 1h at 100V. The biorad system contained a cooling unit (ice). Transfer of proteins was confirmed with Ponceau S Blocking in PBS, 0.1% tween-20, 5% milk powder for 1 hr at room temperature SECONDARY ANTIBODY ECL Anti-rabbit IgG (Amersham NA934V) 1:5000 dilution in corresponding buffers 1hr at room temperature, shaking Wash steps: 3 times 10min in corresponding buffer HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? nitrocellulose or PVDF membranes antibody concentration (2-4 ug/mL) pH during antibody decoration of membranes (pH 7-8) Tween-20 concentration (0.05-0.1%) ADDITIONAL NOTES The sequence of the synthetic peptide used for raising the antobody is present in our mRFP protein sequence This RFP detection on mRFP was a test experiment. I like to use these antibody for mRFP-tagged proteins. Faint bands and aspecific bands are therefore not desired. I added a detection of one of the experiments

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Answer

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. The results that you have been obtaining are most strange. You are clearly detecting a product in your negative control cells of a similar size to that of your RFP tagged protein. Given that this antibody is a polyclonal it is understandable that there is a degree of reactivity against proteins other than that raised against. However, the degree of reactivity with the protein at a size equivalent to your RFP tagged protein is most strange. To fully determine whether the cross reactivity of this antibody is indeed the results of cross reactivity by the antibody I would like to recommend that you perform a no primary control experiment. Often the non-specificity of the secondary antibodies is discounted. However, our lab team often find some secondaries demonstrate non-specificity and non-specific bands attributable to poor specificity of the secondary antibody. Should a no-primary control experiment result in a clean blot demonstrating that this reactivity is attributable to ab28664 please get back in touch with me an I will be happy to provide you with a replacement antibody or credit against your purchase (provided your purchase was made within the past 90 days).

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11-19 of 19 Abreviews or Q&A

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