How to get rid of DNA from sample of western blot?
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:
>>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
>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
> 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?
>The Murdoch Institute,
>R.C.H. Flemington Rd, Parkville, 3052,
>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.
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
hope this helps!
Internal Med.I,Dept. Oncology
University of Vienna
E-Mail:a8803349.nospam at unet.univie.ac.at
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