IEF unit recommendations.

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Wed Jun 9 00:30:32 EST 2004


"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.
 
> 
> 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.


> 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?




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