Monomeric Abeta detection in brain of APP tg mice using western blotting

Stanley Cheung via methods%40net.bio.net (by stanleycheungkk from yahoo.com)
Sat May 16 00:55:05 EST 2009


Hi there,

Does anyone have experiences in detecting and quantifying soluble monomeric Abeta in brain of APP transgenic mice (Tg2576). There is no clear description on the loading amount in journals. I tried many times and eventually found a paper described using 100ug to 250ug of brain homogenate in TBS after ultracenfriguation, 100k xg. However, I believe there is a limited total protein concentration which can be extracted using TBS during homogenization. Actually, 250ug sample means too large volume for loading on mini-SDS-PAGE gel. If for detecting monomeric Abeta (~4KD), the volume of the loading sample should be very small for better resolution. I tried to use TCA precipitation to reduce the volume, but the monomeric Abeta disappeared and the precipitate cannot be dissolved completely. Compared with samples without ultracentrifugation (using the sample TCA precipitation method) and the pellet obtained after ultracentrifugation, monomeric Abeta can be seen
 in these samples on the blot. It makes me to think that TCA precipitation causes too much protein loss for small peptide or the abundance of the peptide in the sample is too little. I should not use the whole brain for doing western blotting of Abeta, it consumes too much sample. I found most papers only use ELISA for soluble Abeta detection, or immunoprecipitate the Abeta from large amount of sample and load it on the gel for WB. However, these two methods are too expensive in my lab. Lyophilization doesn't work too, because it increases the salt content too much which distorts the bands in the gel and the lyophilized sample cannot be totally dissolved in reduced volume. Can anyone suggest the loading amount and ways to concentrate the sample for WB detection? Or is it practical to use WB for such detection?
Thank you very much for your opinion in advance.

Sincerely,
Stanley

PhD Candidate
Department of Medicine and Therapeutics
Faculty of Medicine
Chinese University of Hong Kong

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