Western Blotting of Amyloid Beta - (Apr/25/2013 )
I am working on the C. elegans neurodegenerative model. I am doing drug analysis against them. When I do do its western blot by using sigma monoclonal antibody at different conc. I don't get my desired bands at 4kDA. If I increase the conc of primary antibody, it gives a alot of non specific bands.
If have also used 4G8 antibody but no luck.
Can any body help me in this? Your suggestions are highly welcomed
have you followed the procedures posted by covance (this link will download a pdf of the protocol)?
4g8 (and 6e10) were developed at my institute. i've used it and found it to be finicky about the conditions used (although we used it with a higher salt concentration (500mM) than covance specifies to improve specificity).
covance also gives specific information on how much to use.
Maybe there isn't enough a-beta being produced by your c-elegans, I use mouse models and mine have always worked but obviously mouse brains produce more a-beta. Try using a stronger ECL agent that can amplify your signal, or use a secondary antibody that will amplify your signal. Not sure how much funds you have, but you could use biotinylated antibody, then use streptavidin/HRP conjugate.
you need a control stain to show separation of proteins at your size of interest