Overview

  • Product name
  • Description
    Rabbit polyclonal to V5 tag
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ChIP, IP, WBmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide corresponding to Parainfluenza virus 5 V5 tag aa 50-150 conjugated to keyhole limpet haemocyanin.
    Database link: P11207
    (Peptide available as ab15829)

  • Positive control
    • E. coli Positive Control (Escherichia coli ) Whole Cell Lysate (ab5395) can be used as a positive control in WB. This antibody gave a positive signal in Western Blot when tested against a V5-tagged protein.

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

Our Abpromise guarantee covers the use of ab15828 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
ChIP Use at an assay dependent dilution. ChIP was performed with 25 ug chromatin, 5ug of antibody and 20 ul of Protein A/G beads.
IP Use at an assay dependent dilution. PubMed: 20850014
WB Use a concentration of 0.5 µg/ml.

Target

  • Relevance
    The V5 epitope tag is derived from a small epitope (Pk) present on the P and V proteins of the paramyxovirus of simian virus 5 (SV5). The V5 tag is usually used with all 14 amino acids (GKPIPNPLLGLDST), although it has also been used with a shorter 9 amino acid sequence (IPNPLLGLD).
  • Alternative names
    • GKPIPNPLLGLDST epitope tag antibody
    • GKPIPNPLLGLDST tag antibody
    • Protein Rev antibody
    • Regulator of expression of viral proteins antibody
    • rev antibody
    • V5 epitope tag antibody
    see all

Images

  • A stably transfected 293T human cell line harbouring the GAL4 upstream activation sequence was transiently transfected with a V5 or T7- tagged GAL4 DNA Binding Domain construct. 48 hours post transfection Chromatin was prepared according to the Abcam X-ChIP protocol. The ChIP was performed with 25 ug chromatin, 5ug of antibody and 20 ul of Protein A/G beads. A non-specific antibody was used as the negative control. The immunoprecipitated DNA was quantified by real time PCR (SYBR Green approach).

References

This product has been referenced in:
  • Dewari PS  et al. An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein. Elife 7:N/A (2018). Read more (PubMed: 29638216) »
  • Hasegawa A  et al. Role of Fibulin 3 in Aging-Related Joint Changes and Osteoarthritis Pathogenesis in Human and Mouse Knee Cartilage. Arthritis Rheumatol 69:576-585 (2017). Read more (PubMed: 27780308) »
See all 27 Publications for this product

Customer reviews and Q&As

1-10 of 11 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293T cells cultured in DMEM+10% FBS)
Gel Running Conditions
Reduced Denaturing (15% pre-cast Bis-Tris gel)
Loading amount
20 µg
Treatment
H3.3 with V5 tag over-expressed in pTRIEX 3 plasmid. Harvested 48h post-transfection
Specification
HEK293T cells cultured in DMEM+10% FBS
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Dr. Ranveer Jayani

Verified customer

Submitted Dec 19 2017

Question
Answer

I am sorry that this product has been giving results which have made analysis difficult. I am happy to provide the blocking peptide for this product free of charge, so that you might run an immunogen blocking experiment to determine specificity. I have also included of immunogen blocking protocol for your review.

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Answer

I'm sorry but there is no information available regarding how many V5 tags were incorporated into this GAL4 fusion protein.

Please contact us againif you have any further questions.

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Answer

Thanks for your enquiry.

The scientist who may have further information regarding the number of V5 tags on the vector is out of town until next Monday. I will follow up with you early next week.

Thanks for your patience! Please feel free to contact me if you have any further questions.

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Answer

Thank you for your inquiry.
I would like to reassure you that both antibodies are tested and guaranteed for CHIP.
I can confirm that ab15828 was tested by our lab in house for CHIP and therefore I would recommend to use this antibody.
If you find the publications for ab9116 more convincing, I would like to confirm again that this antibody is also guaranteed for CHIP by our Abpromise.
ab15828 has more than one CHIP publication and therefore we believe that it is also valuable information if a publication states that this antibody can be used as a negative control for other antibodies.
I hope this information is helpful. Please do not hesitate to contact me again with any further questions.

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Answer

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for one vial of ab15828. 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 wish you the best of luck with your research.  

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Question

I have recently been in touch regarding the Abcam anti-V5 polyclonal antibody Ab15828. Please find attached a very similar figure from before, showing a western blot of mock (lane 1), the mMsl1-V5 (lane 2), as well as another fusion mMsl1-HA (lane 3), in HeLa cells. Approximately 30ug is loaded in to each well. Lanes 1-3 were tested with a Sigma anti-Msl1 antibody, and lanes 4-6 with the anti-V5 antibody. The former is a peptide antibody that is not very specific, but does recognise our recombinant protein. I blocked the blot in BSA first, and then stripped and re-blocked in milk (both figures are attached). As you suggested the expression of our fusion construct was much better in HeLa cells compared to 3T3. I found very little difference when blocking with milk or BSA. There are definitely two strong non-specific bands between 37 and 50KDa – which we saw before, and certainly not the single band shown in the figure on the Abcam website. I have not tested the 0.2%tween in our washing buffer, but I am sure you will agree it will not get rid of the two non-specific bands mentioned. As explained in my previous email, we are looking for a very specific antibody to use in ChIP, and hopefully for ChIP-seq or ChIP-chip studies. As you might expect therefore, we are fairly concerned by the non-specific bands seen in the Western. Please could you send me your thoughts and the best way to proceed, and whether it would be possible to get a refund or exchange the antibody as suggested? Many thanks and best wishes,  

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Answer

Thank you for letting me know the progress. It does look like the expression in the HeLa cell line is significantly better and the background staining does appear to have been reduced considerably. I can however understand your concern in continuing to perform ChIP. I think it would be worthwhile to perform a "no primary" control to check the specificity of the secondary antibody if this has not been done already. Is the same anti-rabbit secondary antibody being used for the detection of ab15828 as well as the Sigma antibody? The strong non-specific bands at 40, 45 and 60 kDa as well as the weak non-specific band at 30 kDa appear to be present in both blots and may therefore be as a result of the secondary antibody. This non-specificity could be reduced by the addition of Tween to the dilution buffers as well as the blocking buffer used, and reducing the concentration of the secondary antibody used. I would also suggest now that you are seeing a strong signal, reducing the amount of lysate loaded to 10 µg per well and increasing the dilution of the antibody to 0.25 µg /ml as well as attempting the incubation at 4°C overnight instead of 1 hour at room temperature. This may again increase the specificity seen further. However, if you do not think the secondary antibody could be contributing to the non-specificity seen I can suggest an alternative as a replacement, ab9116. This antibody has also been used for ChIP with V5. More information can be found from the following reference:   Jagani Z et al. Loss of the tumor suppressor Snf5 leads to aberrant activation of the Hedgehog-Gli pathway. Nat Med : (2010).  PubMed: 21076395 If this alternative is not suitable I can also offer a refund. I look forward to hearing your thoughts.  

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Question

Dear Scientific support, Many thanks for your reply regarding my troubles with the anti-V5 Ab15828 antibody. Please find attached the questionnaire that I’ve filled in showing the details of our Western Blot protocol. Please also find attached the image of my blot. The lanes are as follows: 1: 3T3 lysate transiently-transfected with empty pcDNA3 (negative control), Sigma anti-MSL1 HPA022800 2: 3T3 lysate transiently-transfected with pcDNA3 containing the mMsl1-V5 construct, Sigma anti-MSL1 HPA022800 3: 3T3 lysate transiently-transfected with empty pcDNA3 (negative control), anti-V5 Ab15828 4: 3T3 lysate transiently-transfected with pcDNA3 containing the mMsl1-V5 construct, anti-V5 Ab15828 Please note that the blot was separated for an anti-Msl1 antibody and then the anti-V5 antibody. The Sigma anti-Msl1 antibody also seems to give many non-specific bands, but the expressed mMsl1-V5 can also be seen (arrows). It is a polyclonal antibody raised against peptides. We know by silver staining following protein complex purification from HeLa that hMsl1 runs at approximately 75KDa, and so with the V5 tag this is presumably the band of correct size. Please also note that although I have loaded 30ug protein in to each lane, I was initially unable to detect the result with a BioRad ChemiDoc XRS, and instead had to use Kodak BioMax MS film – which is very sensitive. Because of this I don’t think it is a matter of loading too much protein. There seems to be a huge cross-reaction with proteins just below 50KDa in the lanes 4 and 5 by the anti-V5 antibody. Your help and advice would be greatly appreciated. As discussed on the phone, if we decide it is a problem with the antibody we would be very interested in trying the mouse monoclonal antibody Ab27671. I chose the polyclonal over this antibody as this antibody is claimed to be chip-grade. Many thanks again, please let me know if you need any more information, Best wishes,

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Answer

Thank you for taking time to complete our questionnaire. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. Having reviewed this case, I would like to offer some suggestions to help understand the problems encountered with ab15828. I would also appreciate if you can confirm some further details. I understand you are expressing the protein from pcDNA transfection of 3T3 cells and that purification of hMsl1 from HeLa cells has been performed. Has a similar purification of the Msl1-V5 fusion also been purified? It seems both the Sigma antibody and the one from Abcam are struggling to pick out the band expected at ~80kDa. I am just wondering if this is because the expression levels are very low. This is supported by the fact you had to perform imaging using the Kodak BioMax film and the difference between the positive and negative expression seems weak. If this is the case, it could be confirmed by using the purified protein (if you have any), to test both antibodies, as well as quantifying the level of expression in the 3T3 cells by comparison with the known quantity of fusion protein. May I ask which pcDNA construct you have used? This can have a significant effect on the expression levels from cell line to cell line (Invitrogen reported barely discernible expression with CMV (pTracer) promoter in 3T3 cell line). It may be beneficial to try the same experiment that you have performed but with HeLa cells transfected. I would also suggest carrying out a no primary control, to find out how much of the non-specificity is due to the secondary antibody. Trying an alternative blocking agent (eg milk), adding a mild detergent such as Tween 20 (0.2%) as well as incubating with the primary antibody overnight at 4 degrees may also provide cleaner results. Should the suggestions not improve the results, please do let me know. In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement (with a different batch of this antibody, or with an alternative antibody such as ab27671), credit note, or refund. I hope this information is helpful, and I thank you for your cooperation.

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Answer

Thank you for contacting us. I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. As discussed, I am attaching our questionnaire so that I can gather further information regarding the samples tested and the protocol used. Once I have received the completed questionnaire, I will look at the protocol and see if there are any suggestions I can make that may improve the results. As discussed, attaching the file of the Western blot image would be very helpful. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, I would be happy to replace or refund the antibody. I look forward to receiving your reply.

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1-10 of 11 Abreviews or Q&A

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