Problem with inversed binding on a dot blot

kramerse at wmvx.lvs.dupont.com kramerse at wmvx.lvs.dupont.com
Tue Sep 13 07:48:06 EST 1994


In article <dwhite-2608941328290001 at moe.fcs.uga.edu>, dwhite at hestia.fcs.uga.edu (doug white) writes:
>   I recently ran across a strange problem on a dot blot I was running.
>The autoradiograph image was inverted.  The probe bound to everywhere on
>the membrane except the dots.  It's as if there where alot of non-specific
>binding with the membrane (Genescreen plus) and the dotted RNA prevented
>it. It was a commercial cDNA actin probe.  It was hybridized at 52 C in a
>phosphate buffer, containing 0.5M Na, 50 % formamide and 7 % SDS.  Does
>anyone have an idea what could have caused this? Thanks in advance.
>
>Doug White
>dwhite at fcs.uga.edu

You don't say whether or not you are using radioactivity or
chemiluminescence.  This effect is seen with both methods.
If you are using chemiluminescence, you need to dilute your
probe, primary antibody or HRP-conjugate.  There is too much
HRP on the membrane. This gives you the high background. The
even higher concentration of enzyme on your spots is exhausting
the substrate before your can get the film on the membrane.
This also applies to AP systems.  
If you are using radioactive probes, you need to dilute the probe in
your hybridization buffer and wash longer(or change the buffer).

I hope this helps.



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