Anti-CETP antibody (ab19012)

Attached please find the image of the WB film. (Or do you mean you would like to see the image of the stained gel?)

Thanks for your help

ANSWER:

Thank you very much for forwarding your Western blot image.

The first three samples make a lot of sense to me. The protein is secreted, so I would expect to see much more protein present in serum than in cell or tissue lysates. As I said before, it may be that the protein in your samples is more highly glycosylated than the samples tested (rabbit liver is shown on the datasheet). Also, apparent molecular weight can vary from lab to lab due to differences in reagents. This wouldn't account for such a large difference by itself, but in this case, it may be contributing. In addition, the band above ~50 kDa could be the unglycosylated protein.

Again, for the purified protein, I would load much less, maybe try 10 and 100 ng instead of microgram amounts.

You may wish to play around with the incubation and blocking conditions as well, such as diluting out the antibody further but also removing the blocking agent from the primary antibody solution.

Please let me know if my suggestions helps, and do not hesitate to contact us if you need any more advice or information.

It is curious that ab19012 reacts with rat CETP as neither rats nor mice have plasma CETP activity or a functional CETP gene (e.g. Comp. Biochem. Physiol. 135 (2003) 71-81).

ANSWER:

Thank you for your enquiry regarding ab19012 and your interest in our products.

There are controversial data regarding whether mouse and rat have the CETP activity or proteins. We have used the mouse intestine and rat kidney, both of which show the specific 70 kDa bands.

I hope this information will be useful for you.

When this antibody was tested in Western blotting, how was the sample prepared (the positive control lysate), how much protein was loaded onto the gel, and was a nitrocellulose or pvdf membrane used?

ANSWER:

Thank you for your enquiry. Ab19012 was indeed tested using rabbit liver tissue lysate, and a PVDF membrane was used. The lysate was prepared by directly sonicating the tissue pieces in SDS-PAGE sample buffer. 12% gel was used and 30 ug protein was loaded.

Please contact us again if you have any additional questions.

Does the CETP antibody (ab 19012) work in hamster liver sample for western blot detection? If yes, which secondary antibody is the best choice for western blot detection? May I have detail information about that secondary antibody?

ANSWER:

Thank you for your email. All the information we have on species cross reactivity is specified on the datasheet, these are updated as soon as any new information is brought to our attention.

As far as we are aware, cross reactivity with hamster has not yet been tested for use with ab19012. Should you decide to go ahead and purchase this product, please let us know how you get on by submitting an Abreview and in return we will award you 50 Abcam Points, which can be redeemed on a number of rewards (a further 100 points will be offered for an image).

At the bottom of the online datasheet for ab19012 is a list of compatible secondary antibodies. A suitable secondary antibody to use with this primary for Western blotting is ab6721 - Goat polyclonal to Rabbit IgG H&L (HRP).

Please let me know if you require any further assistance.