Anti-S6K1 antibody (ab9366)

Thank you for contacting us.

Yes the customer would be eligible for a testing discount for ab12864 if they are willing to test it in mouse and/or sheep samples with western blotting. The sequence surrounding the Serine 235 and 236 of human RPS6 (SwissProt http://www.uniprot.org/uniprot/P62753), against which the antibody was raised, shares 100% homology with the mouse protein (SwissProt reference http://www.uniprot.org/uniprot/P62754) and we would therefore expect it to react. I was unable to find a sequence for the sheep protein but it seems that the sequence is highly conserved in mammals and I would therefore expect the antibody also to be able to detect the sheep protein.

In order for me to issue a testing discount could you please provide me with the email address and institute that the customer is with.

I look forward to receiving your reply.

Vielen Dank für Ihre Anfrage und dafür, dass Sie sich die Zeit genommen haben, unseren Fragebogen auszufüllen. Es tut mir leid, dass Sie Probleme mit unserem Antikörper hatten. Ich habe gesehen, dass Sie schon mit meiner Kollegin bezüglich des ab2571 in Kontakt waren. Haben Sie das Problem mit dem ab2571 gelöst? Wir würden uns sehr freuen zu hören, wie Sie vorangekommen sind.

In Bezug auf den ab9366,möchte ich gernedie folgenden Punkte ansprechen und Vorschlägezur Veränderungdes Protokolls machen.

1.) Können Sie bestätigen, dass der sekundäre Antikörper ansonsten gut funktioniert auf diesen Zelllysaten? Gibt der sekundäre Antikörper auch Hintergrundbanden?

2.) Ich kann Ihnen empfehlen, die Zelllysate in einem RIPA Puffer herzustellen. Die Wahl des Puffers ist wichtig, da einige Puffer gewisse Proteine nicht genug gut in Lösung bringen können. RIPA ist ein starker und guter Puffer. In unserem Onlineprotokoll haben wir auch einen Absatz über die Wahl der Puffer: https://www.abcam.com/index.html?pageconfig=resource&rid=11379#A1

3.) Ich kann auch empfehlen, Inhibitoren von Proteasen den Lysaten hinzufügen.

4.) Ich kann auch empfehlen, die Proben zu reduzieren. Falls wir keine spezielle Notiz auf dem Datenblatt haben, wurde der Antikörper unter reduzierten Kondition getestet.

Bitte lassen Sie mich wissen, ob ich Ihnen helfen konnte und zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

Wie Sie wissen,garantieren wiralle Produkte so zu funktionieren wie auf dem Datenblatt beschrieben. Falls wir also das Problem nicht lösen können, werden wir Ihnen diesen Antikörperersetzen.

Ich freue mich wieder von Ihnen zu hören.

Thank you for providing those details.


I can now confirm the discount code is xxxxx for which the expiration date is the 24th of July 2012. In order to activate the code, please submit an Abreview for ab12864 using mouse and/or sheep samples with western blotting, quoting the discount code in the "additional comments" section. More information on this offer can be found from the link:

https://www.abcam.com/collaboratordiscount

If you have any further questions, please do not hesitate to contact us again. I wish you all the best with your research.

Thank you for getting back to me.

I understand your concerns over the antibody. I suggested that the antibody may be at fault not because we have had other complaints from customers but because of the optimisation having been performed by the customer and the highly non-specific blot obtained. However, if you have used a further two antibodies to this target with similar results it may be that the samples do not contain the protein as expected. It may be if they have been stored at -80 for a long time, or gone through any freeze thaw cyclesthat they have degraded with time. It may be worthwhile to test the antibodies used with a positive control such as 293T cell lysate or jurkat cell lysate if you have these to hand? If you do not I would be able to provide this for you.

If you would prefer to take a replacement antibody, I can have this organised too. I have had a look at the antibodies suggested against RPS6. Both ab40820 and ab5961 seem like good choices against the unphosphorylated target. These antibodies have both been validated in western blotting and with mouse samples and would therefore both be covered by the Abpromise. From the antibodies chosen against the phosphorylated RPS6target, ab65748 and ab12864, I would suggest ab65748 would be the most appropriate to use as this has been validated with mouse samples which ab12864 has not (we can only say that we would expect it to work due to the sequence homology of the mouse protein with the immunogen used but we cannot guarantee it).

For all of the antibodies suggested, nonehave been used to detect the sheep protein and I cannot therefore say whether they would besuitable for performing western blottingwith thesesamples.

I hope this information has been of help. I look forward to hearing how you would like to proceed.

Thank you for taking time to complete our questionnaire and for providing me with that extra information. 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. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.

Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial. I apologize for the inconvenience has caused your customer and am pleased to offer you a free of charge replacement, credit note, or refund in compensation. Unfortunately, we only have the same lot of ab9366 in stock at the moment. I would therefore suggest https://www.abcam.com/S6K-antibody-6B2-ab119252.htmlwhich is a mouse monoclonal or https://www.abcam.com/S6K-antibody-ab47504.htmlwhich is a rabbit polyclonal. Both have been validated in WB and with mouse samples.

I would still be veryinterested in seeing the "no primary" control result, just to see if some improvements could be made in improving this non-specificity..

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results. We take customer complaints very seriously and investigate every antibody we feel may not be performing as expected. 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 was hoping that you would be able to confirm the following details:

1. Could you share an image of the blot? This is hugely helpful in understanding the problems you have been experiencing.

2. You have mentioned that the antibody has been stored at both 4°C and -20°C. How was this performed, was it stored initially at 4°C then aliquotted to -20°C? From which storage conditions was the antibody used? Could you share images with me of the antibody used after storage at 4°C and when stored at 20°C?

3. You have mentioned that a "no-primary" control was performed. What was the results of this experiment? If possible, could you share an image of these results?

4. Could you share some more details of the secondary antibody used, the catalogue number would me most helpful.

5. Is the membrane being used fresh or has it been probed with a different antibodythen stripped? If so could you share the protocol used with me?

These details will allow me to more fully understand what may becontributing to the problems observed andfor me to make a more informed suggestion to you as to which antibody or protocol may work better for your samples.

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.