Hybond N Problems
mgruidl at com1.med.usf.edu
Sat Aug 17 16:50:15 EST 1996
Just thought I would pass along a little bit of info.
Amersham has had to change the way they make Hybond N. Unfortunately, this may give
some problems with background. Amersham readily acknowledges that about 30% of their
end users have experienced a problem. I was part of that 30%.
I had routinely used a pre-hyb of 2x denhardt's, 5x SSC and a hyb mix of 2x denhardt's,
5x SSC, 0.3% SDS and Salmon sperm. No problems with the old formulation but every blot
with the new membrane gave a tremendous blotchy background which could not be removed by
stringent washing (0.1% SSC, 0.1% SDS at 65oC). I stripped the membranes in boiling
0.5% SDS but the background is still present.
Another co-worker in the lab uses a different hyb solution which includes pyrophosphate
and he does not have a problem. I reprobed one of his blots with my standard protocol
and I also had no problem.
For the record, Amersham now recommends using the Church buffer recipes for
hybridizations. I have just done another set of Northern blots following their advice.
I'm just guessing, but I believe that the increased amount of negative charge from the
SDS and the phosphate should prevent the non-specific binding of my probe to the "bad"
spots on the membrane.
I would be interested in getting some feedback from others who might have had similar
problems and how they managed to correct them.
I would also be interested if anyone else is interested in developing a specific forum
for posting 'verifiable' problems with any companies product. I have had several
incidents where a product was purchased and the company does nothing to warn the end
users of potential problems. I would assume that would be a bad policy to call
attention to any defect in a product. I don't see anything wrong though with the end
users calling others attention to those defects. It might improve or get rid of those
selling bad products (like oligos and antibodies, just to name two of the worst
offenders). Any reputable company will probably welcome the spotlight. What do you
All of these opinions are mine, not that anyone would want any of them.
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