BLOCKING SLN FOR DIG-SYSTEM!!

Klaus Salger salger at wap18.zi.biologie.uni-muenchen.de
Mon Nov 28 12:36:40 EST 1994


chengs1 at jaguar.uofs.edu wrote:
: A big problem in my work of Southern with Dig system: weak signal and
: blak
: background. Sometimes only 30 min exposure immediately after loading
: Lumiphor530. I tried different concentration of probe but no help. I
: think it's
: the problem of BLOCKING. What is the optimal blocking solution? Any
: sugestion?
: Thanx.

: Shuqiu Cheng
: CHENGS1 at JAGUAR.UOFS.EDU

The original Boehringer reagents should work well with Southerns.
But what about your membrane? Engler-Blum et al. (see below) recommend
positively charged nylon membranes like Hybond N+ (which I use) because
they found bigger lot to lot variations with uncharged membranes.
The reference is: Engler-Blum et al. Anal.Biochem. 210, p.235 (1993)
The authors describe a protocol which allows higher sensitivity by
decreasing the background of DIG-Northerns and DIG-Southerns.
Among other factors they tested different blocking solutions and found
Boehringer's blocking reagent in maleic acid to be best.
Hope this helps
  Klaus

--
Klaus Salger                phone : ++49 (0)89 5902 -502
Zoologisches Institut       FAX   :                 -450
AG MacWilliams              e-mail: salger at zi.biologie.uni-muenchen.de
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80333 Muenchen
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