Membranes, swapping, characteristics

R. Rex Denton DENTON at biomed.med.yale.edu
Tue Jan 9 14:42:24 EST 1996


In <DKq6IE.4GJ at ncifcrf.gov> pnh at cockleberry.ncifcrf.gov writes:

> Last year I posted a message warning netters about a membrane SWAP done by
> Tropix with their Tropix Chemiluminescent detection kit called Southern Light.
> For more information about what happened and the response of Tropix, see the
> article below or you can search the archives for my posting to find out what
> the problem was. 
> 
> Recently, I did a side-by-side comparison of six different membranes using the
> same kit and my proven protocol of semi-dry transfer. The results follow:
> 
> Membrane                               Result
> --------                               ------
> 1. The "Old" Tropilon Plus Membrane     ****    Excellent Membrane
> 2. The "New" Tropilon Plus Membrane     -       No Bands, Not Acceptable
> 3. BioRad Zeta Probe                    *       Total loss of some bands
> 4. BioRad Zeta Probe GT                 **      Spotty bands
> 5. S&S Nytran Plus                      ***     Some spotty bands
> 6. MSI Magna Charge                     ****    Best Membrane (1st choice)
> 
> **** = Excellent
> ***  = Good
> **   = Okay
> *    = Poor
> -    = Don't waste your time
> 
> *******************************************************************************
> * Paul N. Hengen, Ph.D.                           /--------------------------/*

I have had some similar experiences with manufacturers swapping membrane
suppliers,in particular the Schleicher and Schuell switch some years back
where they stated making their own charged nylon membrane instead of selling
the Nytran they had licensed from MSI.  I recently was doing some
northern analysis using BM's Genius/Dig/Chemiluminescent system and getting
nowhere using Downward alkaline transfer and the Hybond Membrane.  I did an
anal retentive type of experiment comparing transfermethods and membranes and
found very disparate RNA retention and hybridization
efficiencies on the membranes that was dependent not only on the membrane,
but the method of transfer but the size of the message as well!
I finally stated using the BM membrane recommended by BM (Isuppose I should
have taken them at their word) and it turned out to be the most suitable
membrane (no commercial endosement intended).    

My question is this:  What types of qualities govern efficient binding of
nucleic acids to these membranes.  I woulds really like to know more about them
based on this experience.

Incidentally, FYI,I have noticed that only a small fraction 
of the nucleic acid appears to really stick to the membrane regardless of
fixation techniques (as measured by methylene blue staining before and after
hybridization) and it would appear that while it is important to optimise the
fixation procedure to elicit the highest S/N ratio, most of the potential
signal simply comes off the membrane, which is really too bad, because it would 
nice if it would all stick to the damn thing!!




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