Hi
i am wondering if anyone has any experience working with the Agilent 3100 OFFGEL Fractionator? i was previously using this for isoelectric focusing of 24cm pH 3-10 non-linear IPG strips from GE. the method used was focusing: total 64kVh, total 8000V, total current of 50uA, total power of 200mW and held at 500V, 50uA and 200mW.
i am looking for a method to focus 18cm pH 3-10 non-linear IPG strips from GE.
Thanks
Hi Shauna,
before I can answer your question I would need some more info.
Do you like to focus proteins or peptides?
Do you prepare to focus in an OffGel mode or with a standard IPG IEF mode?
Best,
Murat
Murat Eravci
Hi Murat
i will be focusing protein. i rehydrate passively overnight and focus using standard IPG IEF mode?
Thanks
Shauna
Hi Shauna
there are detailed informations concerning the IEF Settings for each IPG gel type on GE-Healthcares brochure which you can find on top of the pack that you have ordered. The Settings of approx. 60kVh seems fine but you might need to adjust the different increasing voltage steps in relation to your samples properties. Are you doing the passive rehydration with the OffGel wells or do you use a separate rehydration tray? I am asking because if you use the OffGel wells you can also do an active rehydration which can improve the Efficiency of the Migration of your sample into the gel. By doing so, you will also get rid of some remaining salt contamination of your sample prior to focusing.
Best,
Murat
hey Murat
i am doing the rehydration in a seperate tray. i am borrowing the equipment from another lab so i only have the basic equipment they have so i can only do passive rehydration. i dont have the offgel wells.
thank you for you help
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