How to get rid of DNA from sample of western blot?

Richard Kerr kerrr at CRYPTIC.RCH.UNIMELB.EDU.AU
Thu Aug 6 20:06:28 EST 1998


At 01:49 7/08/98 +0800, you wrote:
>Hi,
>
>Recently, I was involved in doing western blot on retina of SD rat and I
>found that there was a lot sticky, jelly like structure after boiling
>with TBS with SDS, triton x-100 buffer although I centrifuged it can
>sonicate the supernatant, it still here. How should I do so that I can
>get rid of it? These sticky material gave vertically line rather than
>horizontal band in SDS page or NC membrane.

Hi, try a DNAse predigestion, remembering to add protease inhibitors
(Boehringer sell a lot of them) to block any contaminant proteases in the
DNAse prep.
Alternatively, a few things come to mind.
*       you could be using too much tissue for your volume of solublising
buffer....try less tissue         or more buffer
*       try loading less protein on your gel...you may be overloading
*       your post heading presumes that the sticky stuff is DNA and your
post itself does not                mention that stain that you used to give
the  'vertical line' rather than the 'horizontal          band'.....if this
tissue is straight from the rat, then your problem could be hyaluronan or
proteoglycan in your sample, as both molecules are present at high
concentrations in the         vitreous and associated with the cells lining
the eye.
        Try a Streptomyces hyaluronidase digestion prior to running (for the
hyaluronan) or a         an ethanol precipitation for protein rather than
sugar....although this may not remove the          proteoglycan....

any other thoughts out there?

Richard Kerr.
The Murdoch Institute,
R.C.H. Flemington Rd, Parkville, 3052,
AUSTRALIA.
kerrr at cryptic.rch.unimelb.edu.au
Phone (61) 3 9345 5045.
FAX   (61) 3 9348 1391.
'The most interesting things about vertebrates occur in the neural crest.'
	Peter Thorogood.




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