Problems with 2d minigels

[giasco78]

I' ve some problems with 2DE, using 7 cm long strip (3-10 linear) and 10x10 minigels. My sample is mouse brain and the gels show some orizzontal streckings and small amount of focalized proteins. I revised all protocols but i have again a bad focusing. Reading literature and manuals, in my opinion the problem is on the first dimension but i don't know exactly wich problem i effort.
Help me please

[alanamco]

Olá
Realmente o seu problema pode deve ser na focalização. O seu protocolo já funcionou com strip de outros centÃ*metros??? Por exemplo de 18cm???
Se quiser vc pode me enviar o seu protocolo e eu reviso com você.
O ideal para strip com faixa de pH de 3-10 é fazer a hidratação da strip sem amostra e utilizando DeStreaK.
O meu e-mail é alanacatalan@usp.br

[giasco78]

OlÃ*
Thanks for your answer.
I describe you my buffers and protocols for IEF:

Rehydration buffer: Destreak(1.62ul) - Ipg buffer(0.675ul) - sample - R3, in a total volume 125ul

R3: Urea 5M - Tiourea 2M - CHAPS 2% - Zwittergent

The last protocol used (with "good" results) is the following:

S1-Rehydrate-30V-16h-480Vhs
S2-Step and Hold-50V-3h-150Vhs
S3-S'n'H-100V-3h-300Vhs
S4-S'n'H-200V-2h-400Vhs
S5-S'n'H-500V-1h-500Vhs
S6-gradient-1000V-1h-750Vhs
S7-grad-5000V-2:30h-7500Vhs
S8-S'n'H-5000V-4h-20000Vhs
S9-S'n'H-30V-24h-720Vhs

After the first step i put paper bridge (or pads) at double end of the strip, to remove interferring substance (like salts) that i can't remove with chloroform-methanol precipitation.
Keeping in touch!

[kiki06]

Great stuff here I am also running IEF 2-D gels

What system are you using?

Hope you are ok... i am just starting!

[giasco78]

I use Ettan IPGphor 2 Isoelectric focusing system from GE Healthcare.
Good Luck!