IEF unit recommendations.
seanpat at fcm.uncu.edu.ar
Wed Jun 9 10:27:19 EST 2004
More thanks to the listers for their input.
>"Deanne Bell" wrote:
>> Hi Sean:
>> My lab-buddy doing 2-D has used the multiphor II in the past, but his
>> current favorite is the Bio-Rad Protean IEF system.
>Personaly I have made good experiences with the Pharmacia IGPhor system,
>except that the gel stripes used for IEF are rather expensive. It may be
>possible to replace them with other brands however.
>Note that in my application (denauring IEF of transmembrane proteins)
>10,000 Vh proved insufficient for complete focussing, 25,000 Vh worked a
>lot better. YMMV.
How did you manage that if the power supply is integral to the unit
and rated to 10,000V? Or were the specifications/design different in
the first unit?
>> Why more than you asked for but - I personnally think the really crucial
>> element is the IMAGING:
>> 1. HIGHLY recommend Sypro Ruby for: MUCH BETTER reproducibility; Ruby
>> has better linearity for quantitation; Ruby sensitivity exceeds the mass
>> spec sensitivity.
>> It's costly, but you can re-use the stain at least one time without
>> losing too much sensitivity. And I don't use the recommended volume
>> (recommended 700 ml for a large gel...I use 400 mL).
>IIRC Sypro Ruby is Molecular Probes trade name for nile red, staining
>with this chemical has been described in Daban et al., Anal. Biochem.
>199 (1991) 169-174. Using homemade solutions can cut the costs for such
Thanks for the tip Engelbert. Money-saving alternatives are always
useful (and usually necessary down here>
>> 3. a huge evaluation of all 2DE image analysis software on the market
>> found The two best were Progenesis from Non-Linear Dynamics, and Protein
>> Mine from Scimagix.
>> PDQuest and Melanie are much cheaper, but not very good. DeCyder from
>> Amersham analyzes the multiplexed images of CyDyes. It's based on
>> Progenesis and it's fantastic. You can even look at rotating 3D graphs
>> of each individual spot. It's also got all sorts of bells and whistles
>> as far as statistical tools.
>How did the free NIH-Immage perform in this test?
I'd also be curious to know, Deanne, if you tried it. Apart from the
flexibility, NIH-Image and Image J have a great online support
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