Anti-Cryptochrome I antibody (ab54649)

I am happy to accept the offer. I checked the order and lot numbers and they are correct.

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.
As requested, I have issued a free of charge replacement with ab114543 with the order number x.
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.

Dear Abcam technical assistance,

Our research team have bought your antibodies ab54649 and ab119053 against human cryptochrome I and II, respectively. I have done and tested several western blots with varying conditions trying to detect the endogenous proteins from human cell lysates. It appears, as you can see from the image below, that both antibodies detect multiple bands at, and near, the predicted cryptochrome molecular weight of 66 kD. In addition, both antibodies detect a very strong band above 70 kD. Since the strongest band is well above the predicted 66 kD, the result casts a doubt on which bands represent actually endogenous cryptochrome proteins. Cells were also subjected to intense blue light (24h BL), which should elevate the cryptochrome protein levels. However, the treatment had no apparent effect (equal protein loading was verified with Ponceau S staining of the PVDF membrane after transfer).



Since neither of the two antibodies is tested for endogenous CRY proteins in humans, and the fact that blue light experiment did not have an effect on protein levels, further proof of the antibody functionality is needed.



Therefore, I ask you, if you have any control material that you could send me and that I could test, e.g. purified recombinant protein (for antibody competition experiment), or cell extract with reduced or absent cryptochrome proteins?

ANSWER:

Thank you for contacting us.
I am sorry to hear you have been experiencing problems with our Cryptochrome antibodies. The quality of our products is important to us and I would like to reassure you that we investigate all customer complaints.
We have received no complaints about these products and I am sorry that you doubt the specificity. As both Cryptochrome proteins are post-translationally modified for example by phosphorylation, this could influence the electrophoretic mobility of the proteins in the gel. The observed band size may also dependent on other factors such as the buffer system used. For an example see the datasheet of ab93802. Considering this, the bands you observed seem specific to me.
In order to optimise the results and reduce the background staining I would recommend the following:
1) As the cryptochromes translocate to the nucleus it might be useful to analyse the cell fractions cytomplasm and nucleus separately. Protocol details can be found on our website (http://www.abcam.com/index.html?pageconfig=popular_protocols).
2) When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk, if you have not used it already. For unknown reasons some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands.
3) The amount of protein loaded per lane and the concentration of the primary antibody might be optimised, too, as the bands are fairly strong.
As the results appear to be specific and thus the antibodies seem to work, we would usually not send a free of charge control sample. However, in this particular case I am authorised to make an exception and could send you our Cryptochrome I protein ab114543:
http://www.abcam.com/index.html?datasheet=114543 http://www.abcam.com/index.html?datasheet=114543.
We unfortunately do not have a suitable Cryptochrome II protein available, but I hope using ab114543 will help to validate the results.
If you would like to accept this offer, could you please confirm the order details for me so that I could arrange for the shipment?
order number 1018633 (PO135) from 30 Jan 2012
lot number 1230051/GR47664-2 (ab54649) and 1235524 GR71422-2 (ab119053)
I look forward to your reply.