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

Martin Offterdinger a8803349.nospam at unet.univie.ac.at
Fri Aug 7 01:27:47 EST 1998


On 6 Aug 1998 18:06:28 -0700, kerrr at CRYPTIC.RCH.UNIMELB.EDU.AU
(Richard Kerr) wrote:

>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.
>
Hi there
In my experience the best way of removing DNA is to avoid isolating it
together with proteins i.e. using mild detergent like triton x100
together with protease inhibitors, lyse cells or tissue, centrifuge
off the nuclei (not possible if you want to isolate nuclear
proteins!!!) and save supernatant after protein assay.
Other methods like DNAse digestion always bear the risk of substantial
protein degradation.
hope this helps!
martin

Martin Offterdinger
Internal Med.I,Dept. Oncology
University of Vienna
Austria
E-Mail:a8803349.nospam at unet.univie.ac.at
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