IEF unit recommendations.

Sean Patterson 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
>assays significantly.

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 
community.


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