Anti-CIDE C antibody (ab16760)

Thanks a lot for your reply. Yes, the antibody has been ordered by ******, a PhD student in my lab. With my best regards*

ANSWER:

Thank you for confirming those order details. I have processed your request for a refund for ab16760 as it failed to work in western blot as guaranteed. I am sorry about the problems you had with this antibody and if there is anything else I can help you with, please let me know.

Following our last exchange of e.mail (see below), we have made several testes in order to get a specific band with the anti-CIDEC antibody. As you adviced, we have boiled our samples 10 min instead of 5 min; we have blocked our membrane with 5% BSA (in parallel with another membrane blocked with 5% Milk); we have used as positive control human hepatocyte extracts. However, none of these changes improved our results (see enclosed ppt file): we still have many bands (even the GFP in our GFP-transfected cells is recognized by the antibody!). In these conditions, I would be very grateful if you could process a refund corresponding to the price of the antibody ASAP. Could you please let me know how to proceed?

ANSWER:

Thank you for your reply. I am sorry that the protocol tips did not prove to be effective in resolving the problems with ab16760. I am more than happy to process a refund for the original antibody. Before I process this though, I believe I have found your original order but I would just lie to confirm that it was ordered by ******on ***** 2011. If these details are correct, I will go ahead and process the refund.

Thank you for your mail and for your suggestions. I already have a couple of answers to your questions/comments: 1 - Did you use the same loading buffer for both the human and rat samples, as if the loading buffer is old it can cause the ladder effect seen in the human samples. NO, THIS WAS NOT THE SAME BATCH OF SAMPLE BUFFER (ALBEIT THIS IS THE SAME RECIPE)... HOWEVER, I DO NOT THINK THAT WE ARE FACING A PROBLEM OF DEGRADATION OF THE PROTEINS AS THE EXACT SAME SAMPLES HAVE BEEN USED TO BE BLOTTED WITH SEVERAL OTHER ANTIBODIES WITH GOOD RESULTS. IN ADDITION, THE COOMASSIE BLUE OF THESE SAMPLE LOOKS GOOD (NO APPARENT DEGRADATION OF THE PROTEINS) 2-How long did you boil your samples? It could be that these proteins are in a complex and so by boiling for 10 minutes (instead of the usual 5 minutes) this can help break up these complexes and allow the proteins to run at their expected mol weight. SAMPLE HAVE BEEN BOILED FOR 5 MIN. WE WILL TRY BOILING THEM 10 MIN. THIS COULD MAY BE EXPLAIN THE PRESENCE OF THE BAND AT THE WRONG SIZE IN THE RAT COLON SAMPLE. 3-Are you using 5% milk as a blocking agent? If so, switching to 5% BSA may help get rid of some of the non-specific background in the human samples, if the loading buffer is not the issue. WE ARE ROUTINELY USING MILK AS BLOCKING AGENT. WE WILL TRY TO USE BSA AS AN ALTERNATIVE. HOWEVER, I'M NOT VERY CONFIDENT THAT THIS WILL HELP MUCH AS WE DO NOT HAVE A MAJOR BAND, OR EVEN A BAND AT THE RIGHT SIZE. FOR THE CELLS OVEREXPRESSING CIDEC, WE HAVE CHECKED THE PRESENCE OF THE mRNA BY REAL-TIME PCR. THE OVEREXPRESSION HAS THE EXPECTED EFFECT ON LIPID DROPLET SIZE. ALL TOGETHER, THESE DATA ARE IN FAVOR OF THE PRESENCE OF THE PROTEIN. IN THESE CONDITIONS, WE SHOULD BE ABLE TO SEE ONE STRONGER BAND AROUND 30kDa IN THE LANE 1, WHICH WE DO NOT SEE. For all the above reasons, I believe that their is a problem with the antibody. What other positive control (other than rat colon)would you advice me to use in order to check for its specificity?

ANSWER:

Thank you for your reply. I would recommend using either small intestine or liver as another positive control, as both of these tissues have high levels of expression of CIDE C. Please let me know if any of these suggestions helps to generate a band in your western blot experiments. As I mentioned previously, the antibody is covered under our Abpromise and is guaranteed to work in western blot on human and rat tissue. If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund.

Dear Mrs/Mr, We have recently bought an antibody (anti-CIDEC; ab 16760) from your company. However, despite several tests, we could not succeed to obtain a specific band at the expected size. As in the datasheet, the positive control usedfor western blotting was colon tissue lysate, we also tested the antibody on this sample. In these conditions, we could get one major band. However, the size of this band was around 40 kDa (instead of 27 kDa as excepected). Please find enclosed a tif file of the western blot we have done. Lane 1: Hepatoma Huh7 cells overexpressing CIDEC Lane 2: Hepatoma Huh7 cells Lane 3: 10 microgram of rat colon tissue lysate Lane 4: 5 microgram of rat colon tissue lysate What is you opinion regarding this problem? I thank you in advance for your help in this matter.

ANSWER:

Thank you for your patience while I contacted the lab. I was trying to clarify if the antibody would recognize rat tissue or not as there was conflicting information on the website. We have updated the website to show reactivity with rat. As to why you are getting the banding pattern that you are seeing, I am a little unsure as to why you seem to be getting a difference between the rat and human samples, as they have the same molecular weight. Also you are getting a ladder effect in the human samples. I have a few questions/suggestions that may help improve the results: 1 - Did you use the same loading buffer for both the human and rat samples, as if the loading buffer is old it can cause the ladder effect seen in the human samples. 2- How long did you boil your samples? It could be that these proteins are in a complex and so by boiling for 10 minutes (instead of the usual 5 minutes) this can help break up these complexes and allow the proteins to run at their expected mol weight. 3 - Are you using 5% milk as a blocking agent? If so, switching to 5% BSA may help get rid of some of the non-specific background in the human samples, if the loading buffer is not the issue. The antibody is covered under our Abpromise and is guaranteed to work in western blot on human and rat tissue. If we cannot resolve the issue you are having with the antibody then I would be happy to either send a replacement antibody or to process a refund. I look forward to your reply and helping you resolve this issue.

We have recently bought an antibody (anti-CIDEC; ab 16760) from your company. However, despite several tests, we could not succeed to obtain a specific band at the expected size. As in the datasheet, the positive control usedfor western blotting was colon tissue lysate, we also tested the antibody on this sample. In these conditions, we could get one major band. However, the size of this band was around 40 kDa (instead of 27 kDa as excepected). Please find enclosed a tif file of the western blot we have done. Lane 1: Hepatoma Huh7 cells overexpressing CIDEC Lane 2: Hepatoma Huh7 cells Lane 3: 10 microgram of rat colon tissue lysate Lane 4: 5 microgram of rat colon tissue lysate What is you opinion regarding this problem? I thank you in advance for your help in this matter.

ANSWER:

Thank you for contacting Abcam. I am sorry that you are having issues with ab16760 in western blot. I am contacting the lab at the moment to try and see if I can get any information that would explain the banding pattern that you are seeing. I will contact you as soon as I hear back from the lab.