Great separation between 15kDa and 5kDa

[ghalia]

hi,
could someone give me a simple protocole for a Tris-Tricine gel or another protocole for a great separtion between 15kDa and 5kDa.
i would like to note that the Tris-Glycine 15% and 18% with 40 % acry/bis-acry ( 37:1) didn't give me good results.
thank u

[danfive]

Have you thought of using a filter spin column with molecular weight cutoff at 10K?

[admin]

Hello Ghalia,

you can also try a gradient gel from 10%-30%+

Gradient gels can separation anything.

Also there are special gels (tricine? or sequencing gels) that can separate very small differences in molecular weight or kDa proteins.

let us know what worked for you

cheers

[ghalia]

thanks for ur suggestions.
i tried today the gel tris-tricine,but i'm a little bitt worried about the results because the bands are not well determined, they r so much diffuse.
I think because i run the electrophoresis at 30mA for 6h and a half, is it the cause ??
if yes what r the perfect parameters for running a tris-tricine gel ??
thanks a lot
i appreciate any suggestion

[danfive]

Originally Posted by ghalia

thanks for ur suggestions.
i tried today the gel tris-tricine,but i'm a little bitt worried about the results because the bands are not well determined, they r so much diffuse.
I think because i run the electrophoresis at 30mA for 6h and a half, is it the cause ??
if yes what r the perfect parameters for running a tris-tricine gel ??
thanks a lot
i appreciate any suggestion

6 hours and a half for a gel run seems excessive time, your voltage must be very low. Try 100-150V (set no limit or max for mA) for approx 50min, or until tracking dye (bromophenol blue) reaches the bottom.

[ghalia]

hello danfive
thx for ur reply but i would like to pay your attention that i'm running big gels that's why 6 hours seems not so long but maybe u'r right the voltage seems very low, i run yesterday another tris-tricine, for 30 v util the sample leave the sample pocket and than 150 v for 19 h and 200 v for the last 3 hours of my electrophoresis. I get good results, i saw band's markers till 3,5 kDa in commassie Blue G-250 and silver stain. But i notice that the bands of my samples between 15 kDa and 6 kDa are not quite clear !! they still a little bitt diffuse what do u think ??
concerning filter spin column with molecular weight cutoff at 10K, i have tried the ones from millipore Ultrafilter4 but they didn't work well, have u tested a spin column with cut off 10k that works good ? if yes could you give me the name of it and the protocole ??
thx a lot and good luck

[danfive]

I use Millipore Ultrafree-MC 10,000 NMWL Filter Unit Cat no. UFC3BG00. Centrifuge at 5,000g for 30min.