Anti-KCNQ4 antibody [N43/6] (ab84820)

Thank you for your answer and for your cooperation.

I am sorry that the ab84820 required more optimisation than expected and for the inconvenience this has caused.As requested, I agreed to send a free alternative antibody in exchange for the feedback via an Abreview for both antibodies. I have issued now this free of charge replacement with the alternative antibody ab56456 with the order number 1096821.

To check the status of the order please contact our Customer Service team and reference this number.

We look forward hearing back from you andwish you the best of luck with your research.

Thank you for your reply.

We can send you a new antibody in exchange for your feedback, however I am not sure whether this will resolve your question.

I have discussed this case also with a colleague of mine. Indeed, we both think that the results obtain seem to correlate and that with a new antibody we would expect the same results. The reason why we think the results to correspond to what is expected are:
1.) The results improved upon reduction of the heating, which correspond to the expected behaviour of this target protein.
2.) The results show some duplicate band, which would correspond to the two isoforms as described on SwissProt (http://www.uniprot.org/uniprot/P56696),
3.) I agree that the observed molecular weight is not as expected, however,as mentioned in my previous emails, Ithink it would be very usefulto compare the compatibility of the buffer and molecular weight marker. Different molecular weight marker migrate different in different buffer systems. Variations of 15% in the expected molecular weight can be seen. See the images of our MW marker as an example: https://www.abcam.com/Prism-Ultra-Protein-Ladder-10-245-kDa-ab116028.html

Please let me know what you think about these points. So far, we had no other negative feedback on this antibody neither. I can provide you with an alternative antibody if you provide us feedback on both antibodies, used in the same conditions, via an Abreview (https://www.abcam.com/abreviews). We do have several other antibodies against KCNQ4 which have been tested in Western blot and on human cells. Please see here the links to their datasheets:
https://www.abcam.com/index.html?datasheet=56456 (or use the following: https://www.abcam.com/index.html?datasheet=56456).
https://www.abcam.com/index.html?datasheet=65797 (or use the following: https://www.abcam.com/index.html?datasheet=65797).
https://www.abcam.com/index.html?datasheet=82096 (or use the following: https://www.abcam.com/index.html?datasheet=82096).
https://www.abcam.com/index.html?datasheet=110373 (or use the following: https://www.abcam.com/index.html?datasheet=110373).

Thank you very much for your cooperation. Please let me know if you have any questions. I look forward to hear your opinion on the above mentioned points and how you would like to proceed.

I just write you to inquire whether you have yet had the opporunity to look into my comments/suggestions? Please let me know also if you have any questions or concerns - I would be happy to discuss them with you. If the problem has been solved, I would also appreciate if you can let me know, so I can update our records accordingly.

Here is also a link to our online Western blot protocol, where each step of theWestern blotprocedureis described in detail: https://www.abcam.com/index.html?pageconfig=resource&rid=11375
Blocking the membranes with proteins, such as milk or BSA, is very important to reduce the background. Also, changing from one of those reagents to the other one can significantly improve the results (e.g. Western blot image on our datasheet for ab9385 https://www.abcam.com/index.html?datasheet=9385 (or use the following: https://www.abcam.com/index.html?datasheet=9385).).

I am looking forward to hear back from you. Please do not hesitate to contat us also should you have any other question.

Thank you for your reply.

I am sorry for the confusion of the origin of the samples. Indeed, as you are using human samples, there are two isoformes described. One is at 71kD and the other one is at 77kD. Please see also this link to SwissProt for the details: http://www.uniprot.org/uniprot/P56696
As I can not read the molecular weight marker annotation on your Western blot, please check whether this could corrspond to the two bands you are observing on the WB on the right side.

Please check also whether the protein ladder is compatible with the migration buffer. Indeed, depending on the buffer, the protein ladders can migrate at different molecular weights. Please see the images on one of our protein ladders for illustration: https://www.abcam.com/index.html?datasheet=116028 (or use the following: https://www.abcam.com/index.html?datasheet=116028).

When I was asking about the blocking of the membrane, I ment the protein blocking - before performing the immunostaining. Do you not incubate the membranes in a protein solution with either milk or BSA before incubating with the primary antibody dilution? As you are using BSA in the antibody dilution, I would think that you are using also a BSA blocking step prior to the antibody incubation?

Please let me know your feedback. If these points do not solve the problem, I will be pleased to continue to investigate the problem. If the antibody is not working, please be assured that is covered by our Abpromise guarantee to work as stated on the datasheet and we would therefore replace it (if purchased within the last 6 months).

Unfortunately, the laboratory has no other images as the one on the datasheet, nor do we have tubes of the immunogen fragement available. I am sorry.

I am looking forward to hear back from you.

Thank you for taking time to complete our questionnaire. I am sorry that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Thank you very much for the two images as well. Can you explain to me what the difference is between the blots on the left and the blot on the right?

As discussed, I have contacted the laboratory for more information and explaining the problem.The laboratory replied thatit sounds like the band is in the right area, but that a doublet would generally mean a small difference in size.It could be a deletion, or proteolysis fragment.The laboratory has not seen this, but that is not to say it cannot occur, as this isvery much an evolving area.The work of the laboratorywas on rat brain. In summary, the laboratory head thinks thatthe fact to havea doublet could either be due to a preparation artifact, proteolysis fragment or possibly a generic variation, deletion, pseudogene etc.

Indeed, there is no described isoform to my knowledge in mouse, however in human there are two isoformes, one at 77kD and one at 71kD. It might well be therefore, that the mouse isoforme still needs to be discovered? http://www.uniprot.org/uniprot/P56696

In order also to exclude any artifact, have you checked whether any background banding could be due to the secondary antibody? This could easily be checked by running the same samples and the same blot just without the primary antibody.

Can you please also let me know how the membranes have been blocked, have you used milk or BSA? Often changing the blocking buffer can also improve the signal.

Please let me know your thoughts. I am still following up on the possiblity of the blocking peptide and the images and will get back to you in this regards.

As mentioned before, 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, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Thank you for calling us.

As we discussed on the phone, I have contacted thelaboratory for the additional information. I am also attaching the discussed questionnaire so that we can gather further information regarding the samples tested and the protocol used. This will enable us to look with more details into the results you obtained and try to troubleshoot them.

If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I look forward to receiving your reply.

Thank you very much for your email.

I have now received an email from lab. What my colleague has suggested is attached below;

Multiple bands are very common with ion channels in general. Proteolysis is very common – it has to do with liberating the channels from the membranes – the protective barrier against the proteases is lost. What I would suggest is two-fold. The use of proteolysis inhibitor cocktails when preparing the lysate, and secondly, do not boil it or heat to 90°C when prepping it to run on SDS. Keep all conditions the same but then instead heat at 37°C for 15 minutes instead. That worked for us, although the outcome is still of course dependent on the initial proteolysis if there is any. Other than that any western protocol should work just fine. Proteases tend to be very heat resistant (they can work very well at 55°C+), and because (all) the reactions are still temperature dependent for the short time they are active at the high temperature they can inflict a high level of degradation.

I hope this information will be helpful. Should you have any other inquiry please do not hesitate to contact me.

I can confirm that I have send an email to my colleaguein lab and requested the productspecific WB protocol; I will be able to forward the same once I get a reply from them.

Many thanks for having patience.