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PELs subcellular fractionaction

PELs subcellular fractionaction - Protocols and Methods Forum

PELs subcellular fractionaction - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 09-05-2004, 03:48 PM
Jayakumar, R
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Default PELs subcellular fractionaction



Have you tried using isopycnic gradient separation of the cytoplasmic organelles. During differential centrifugation methods, the nuclei is usually easily separated by a 1000 xg spin for 6 min. when removing the cytosolic fluid after the spin, try not to take the cytosol completely out. leave a mm or two above the nuclear pellet. It also looks like your nucleus is lysing while doing the homogenization. What sort of homogenization do you do and with what capacity homogenizer. I always like to use the glass homogenizer with a tight pestle by hand after swelling the cells in an 10 mM hypotonic buffer. I find that more gentle and causes much lesser lysis. I was always more interested in the cytosolic components especially the mitochondria than in the nuclei and must admit have neve checked for nuclear contamination, since it never concerned us. And dont keep cchaning your methods. If a particular method has worked for somebody else, keep practising it, till you achieve the level of perfection that you need to get good fractions. Subcellular fractionation techniques is all about handling and timing, and nothing else. I could suggest a few tricks if you can send me your protocol.
But through isopycnic gradient purification, we have always successfully managed to separate out the nuclear fraction and each of the major organells out like the mitochondria, lysosomes, peroxisomes, golgi, ER etc. If you are interested to try out gradient purificaiton, check out materials like Iodixanol (I love this), or metrizamide (not available very easily), percoll etc. if you need any help let me know.

best of luck
jai

-----Original Message-----
From: [Only registered users see links. ] on behalf of ganter
Sent: Sun 9/5/2004 11:28 AM
To: [Only registered users see links. ]
Cc:
Subject: Re: PELs subcellular fractionaction

[Only registered users see links. ] ("Jayakumar, R") wrote in message news:<97101976F8A044468CA74FE11883B90E07496361@VIS TA.roswellpark.org>...


I wanted isolate separately cytoplasmic and nuclear proteins from
cells. These are human primary effusion lymphomas cells, which are
suspension cell cultures and latently infected by HHV8 (KSHV).The
specific problem is that my cytoplasmic extract is always contaminated
by proteins from nuclei fraction.(has been checked by cytosolic and
nuclear markers, by Western)
I tried many, many different options i.e hypotonic buffers, various
detergent and many variants of salt concetration....

ganter



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