Anti-Melanopsin antibody (ab19306)

I used your antibody for Western Blot with the rat and another diurnal species. At high concentrations of proteins, I have multiple bands appearing , but when I reduced concentrations, there is just one band left : a about 43kDa band. I don't see any 85kDa (only with a pure extract where I see many bands!). Could my 43kDa band be the melanopsin protein?

I want to order a new batch, but I first want to be sure of the specificity of the antibody.

ANSWER:

Thank you for your enquiry. I am sorry to hear you are having difficulty obtaining satisfactory results from this antibody (ab19306).

This antibody has been tested on total protein extracts of HEK293 cells with or without a c-myc tag (to verify the specificity of the antibody). From overexpressed melanopsin, a band is observed at the expected molecular mass (~53 kDa). There us also a larger band (~85 kDa) which is glycosylated melanopsin (this disappears under deglycosylated conditions). The antibody is known to give bands of smaller molecular weight from intrinsically degraded expressed melanopsin protein.

I can suggest that the band you are observing at 43kDa is the non-glycosylated melanopsin. Although this is 10kDa less than the expected size, molecular weight markers are not accurate enough to give a precise molecular weight. The larger glycosylated band at 85kDa has probably disappeared under reducing conditions due to affects of the reducing and denaturing process on the glycosylation. Ensure you are reducing and denaturing adequately so the protein runs at the correct molecular weight.

I hope this information is helpful. However, if you are still concerned about your results, I have enclosed a link to a questionnaire which will help you put your protocol information together very easily so we can look into the details of the experiment and provide some assistance.

http://www.abcam.com/index.html?section=western&pageconfig=technical&mode=questionaire

We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 90 days of purchase), and if it appears that the antibody is at fault, a credit note/refund will be offered.

Please do not hesitate to contact us again should you require further assistance. Good luck with your experiments.