Anti-V5 tag antibody - ChIP Grade (ab15828)

Would it be possible to receive item ab15828 (Anti-V5 tag antibody - ChIP Grade) as a replacement for the previously ordered antibody.

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.  

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,  

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.  

Many thanks for your reply to my inquiry on the Ab15828 anti-V5 antibody. Unfortunately we do not have a purified mMsl1V5 to test the antibody. The construct I am using is the pcDNA3 vector, which does have the CMV promoter. I wasn't aware of the potential problems with the CMV promoter in 3t3 cells, and will definitely express my construct in HeLa cells as you suggest to repeat the experiment. I can also try milk as a blocking reagent, and adding 0.2% tween as you suggest as well. I will let you know how we get on with these approaches as soon as possible, and then we can decide whether there is a need to refund or exchange the antibody. Many thanks again and best wishes,

ANSWER:

As far as I could tell, expression in HeLa cells should not be a problem with the CMV promoter so if you still get the same results after trying this get back to me and we'll discuss what the next course of action is (refund/alternative antibody).

I hope it goes well.

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,

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.

ChIP grade antibody ab15828 used in mouse sampled with transient transfection of V5. Both control and transfected samples show non-specific bands.

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.