Isolating Platelets and removing plasma proteins?
(by nobody from nospam.not)
Thu May 13 20:38:53 EST 2010
dk from no.email.thankstospam.net (DK) wrote in
news:JGTGn.9461$TL5.1130 from newsfe24.iad:
> In article <MPG.2655c5c4f1d310d298968a from News.Individual.DE>, Dr
> Engelbert Buxbaum <engelbert_buxbaum from hotmail.com> wrote:
>>In article <mailman.642.1273703580.25217.methods from net.bio.net>,
>>silverstein.joshua from gmail.com says...
>>> Hi team -
>>> Anyone have a good protocol for isolating platelets and removing
>>> plasma proteins through a Sepharose column? I've been having
>>> trouble with this and fresh blood is hard to come by!!!
>>You can not separate cell typess by gel filtration. This is usually
>>done by centrifugation, either fractionated or in an isotonic
>>gradient. Such gradients can be formed for example with ficoll,
>>metrizamide or nycodenz. Since only cells, but not the plasma
>>proteins, enter such a gradient, this step will also separate the
>>cells from the proteins.
> I do believe platelets separation from plasma on Sepharose CL2B
> is an established protocol. It's not clear what "trouble" above
> actually means. Column clogging? Platelets lysing/activating? Low
I forget the actual kind of Sepharose, but it is an established protocol
to separate platelets from plasma. One can prepare platelet-rich plasma
rather easily be choosing a) the right anticoagulant, b) discarding the
first few drops or ml of blood so as to eliminate any "tissue juice" that
can activate the platelets, and c) the right centrifugation conditions.
I have never done Sepharose separation of plasma and platelets, but it is
a well-known protocol. The difficulty is probably in knowing the exact
volumes to use, and the right diameter tube. I'd contact Dr. Ellinor
Peerschke at Mount Sinai in NY for experimental details.
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