how to see A beta by western blot - (May/15/2008 )

Hi all
I am trying to see abta 40 and 42 by western blot using tricine gels, but still the bands are not clear and most of the time the protein aggregates. Do you know any reliable method or any trick to avoid aggregation and see the monomers clearly?

Thanks soooo much

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how do you prepare your samples for PAGE? is it SDS-PAGE? do you use Laemmli buffer with boiling samples?

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what % gel do you run?

you may want to try a 16.5% separating gel or a 10-20% gradient to keep the bands sharp.

also...

you may have better results with abeta 1-40. 1-42 tends to aggregate more readily.

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Have you seen M&M in this paper?[attachment=4704:8844.pdf]

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I use Laemmi buffer and I run tricine gels 16%. I tried to boil the samples in 6%sds and then pellet the proteins at 45000 rpms, but I lose a lot of protein.
Thanks so much

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is that laemmli sample buffer or loading solution? or electrode buffer?

instead of boiling your samples, try using 65C for 10-20 minutes.

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I am trying to see abta 40 and 42 by western blot using tricine gels, but still the bands are not clear and most of the time the protein aggregates. Do you know any reliable method or any trick to avoid aggregation and see the monomers clearly?

*** Gradient gels are better. Tricks to avoid aggregation : Go for ultra sound water bath, DMSO keeps them as Monomers.

Aß aggregation is something *very* big issue in AD field, and thats how its running..

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