PELs subcellular fractionaction

Jayakumar, R R.Jayakumar at RoswellPark.org
Sun Sep 5 10:48:20 EST 2004


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.=20
    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.=20
  =20
    best of luck
jai

-----Original Message-----
From:	owner-methods at hgmp.mrc.ac.uk on behalf of ganter
Sent:	Sun 9/5/2004 11:28 AM
To:	methods at hgmp.mrc.ac.uk
Cc:=09
Subject:	Re: PELs subcellular fractionaction

R.Jayakumar at RoswellPark.org ("Jayakumar, R") wrote in message =
news:<97101976F8A044468CA74FE11883B90E07496361 at VISTA.roswellpark.org>...
> I have done tens of subcellular fractionation of human cells.  So what =

> sort of cells are we looking at and what level of purity of fractions=20
> are we talking of.  What sort of fractions do you want from the cells? =
=20
> Are they human cells, are they suspension cell cultures or monolayers =
or=20
> tissue samples? Try giving all the information you need before putting =

> up a question like that.  It should help to get help fast.
> Jai
> =20
> > ----------
> > From: 	owner-methods at hgmp.mrc.ac.uk on behalf of ganter
> > Sent: 	September 3, 2004 11:44 AM
> > To: 	methods at hgmp.mrc.ac.uk
> > Subject: 	PELs subcellular fractionaction
> >=20
> > Any useful protocol for subcellular fractionation of cells latently
> > infected by KSHV????
> >=20


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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