Tris Tricine Gels for amyloid-beta Oligomers - (Jun/14/2006 )

I would like to make a 16.5 % tris-tricine gel. I trying to detect oligomers in an Amyloid-beta solution. Can anyone help me to make this gel, i never did it before, Thanks Niek

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what do you need help with? do you need the gel formulation? running conditions?

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I would like to have the gel formulations and especially the sample buffer and the concentration of amyloid beta that i have to put on the gel....Tx Niek

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the formulation of Schagger and von Jagow:

10X anode buffer (+):
2.0 M Tris
adjusted to pH 8.9 with HCl

10X cathode buffer (-):
1.0 M Tris
1.0 M Tricine
1.0% SDS
pH should be 8.25 (do not adjust)

gel buffer:
3.0 M Tris
0.3% SDS
adjust pH to 8.45 with HCl

Acrylamide (49.5%T, 3%C):
48 gm Acrylamide
1.5 gm Bis-acrylamide
dw to 100ml

APS: 10%
0.1 gm Ammonium Persulfate
dw to 1 ml

Gel Formulations:
4% Stack 10% Spacer 16.5% Separation
Acrylamide 1 3.05 10 ml
Gel Buffer 3.1 5 10 ml
Glycerol 4 gm (in separating gel only)
Di Water 8.4 6.95 ~7 ml
TEMED 10 5 10 ul
APS 100 50 100 ul (just before pouring)
(this table is for 3 gel formulations, the forum software stripped out the spacing)

run at 30V for ~1 hr or 50V for 20-30 min (until dye passes through stack)

then at 90 to 100 V for ~16 hr

electrodes '-' (top) to '+' (bottom)

prepare sample as you would for any sds page.

sample load depends on your detection method.

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mdfenko Tx !!!!Have a nice day and i wish all the best! Greetings from the Netherlands, Niek !!

P.S. The USA had a lot off luck in the match against Italy....

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Hi: please note do not let the anode and cathode buffer mix.
have a good day

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I need to separate small molecular weight proteins below 14kDa. I know I need Tris-tricine gels for that. My question is whether stacked or resolved gels are prefereble for the Tris-tricine gels. I am more accustomed to running Tris-HCl stacked gels. I was also going to run it at a constant 125V, any comments or suggestions would help me out, this discussion has been helpful for me too. Thanks

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see my earlier post with the formulation. it includes a stack and a spacer gel as well as the running conditions. that should answer your questions.

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