I am trying to quantify the levels of C-terminal fragments of APP, generated after BACE1 cleavage in APP/swAPP cell lines (in different experimental conditions). I have tried some commercially available antibodies but unfortunately none of these antibodies are functional. Can somebody recommend me (from ones own experience) an antibody which is able to detect CTFs of APP in cell culture models?
Thank you Anushruti for your answer. But is this antibody really functional in your experimental results? Its because of the fact that I have tried three different antibodies (which have been claimed to be able to detect CTFs in Immunoblotting), but I didn't find any significant success. The principal reasons might be due to relatively short life span of the non-phosphorylated CTFs as compared to phosphorylated ones in the membrane microdomains. But in principle, the phosphorylated CTFs should also be detected by an antibody of which immunogen contains almost the entire sequence of CTFs. But thanks again for sharing the information.
Article Membrane-Microdomain Localization of Amyloid -Precursor Prot...
Have you tried the Calbiochem (Merck) Anti-Amyloid Precursor Protein, C-Terminal (751-770) Rabbit pAb (#171610)? With that one you easily detect APPfl, all CTFs and AICD.
If your antibodies are not sensitive enough, I would recommend an enrichment of the CTFs by immunoprecipitation with an APP C-terminal antibody.
Thank you very much Linda, for your kind suggestions. Yes, I have also tried Calbiochem #171610 but unfortunately it is also non-functional.
I have also enriched the APP C-terminal fragments by using tag-specific antibody ( for overexpression, I use APP-YFP construct). But my eventual aim is to compare the levels of phosphorylated and nonphosphorylated CTFs in different conditions by densitometric analysis, which won't be possible without quantifying the total levels of CTFs produced in different conditions.
From my experience, A8717 is sufficient to detect all the APP-CTFs even in endogenous level. Also A8717 is the best among others, for example Chemicon AB5352. There is also good antibody from Cell signaling to detect phos-APP. For western blots there won't be any trouble to detect APP-CTFs in overexpressed system. You should consider to improve your experimental system to increase sensitivities though.
@Lei Liu: Thanks for sharing the information. Actually, I have already purchased A8717 and tested its efficiency. The antibody seems to be quite sensitive and is able to detect not only the CTFs but also the AICD (even though 12% bis-tris gels are being used). But thanks again for sharing the information and all the best for your experiments.
Dear Hassan,
We use the commercial Y188 and it works really nice.
http://www.abcam.com/amyloid-beta-precursor-protein-antibody-y188-ab32136.html
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