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Western Blotting of Amyloid Beta

Amyloid Beta C. elegans Western Blot

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#1 Waqar Ahmad

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Posted 25 April 2013 - 02:22 AM

Hi
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

#2 mdfenko

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Posted 25 April 2013 - 05:08 AM

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.
talent does what it can
genius does what it must
i do what i get paid to do

#3 notjustalabtech

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Posted 09 June 2013 - 11:11 AM

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.

#4 Inmost sun

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Posted 18 July 2013 - 02:52 AM

you need a control stain to show separation of proteins at your size of interest





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