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OPS Diagnostics, LLC
291 Route 22 East
Salem Industrial Park
Building 6
Lebanon, NJ 08833
TEL: (908) 253-3444
FAX: (908) 575-1660
Email:
info@opsdiagnostics.com
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Application Notes >Application
Table
Certain animal
tissues are relatively easy to homogenize and can be processed by a
variety of methods. Liver, thymus, fat, and brain, all soft tissues, can
be homogenized by bead beating, shearing with rotor stators, and by
cryogenic grinding. Softer tissues have less connective tissue than
muscle and thus much easier to homogenize.
The major
considerations in deciding on a homogenization method are sample size and
target analyte. Rotor stators are useful for a limited number of samples
while bead beating is the best high throughput method. For RNA
extractions where RNAlater is NOT used, grinding frozen samples can be
performed with the
CryoGrinder
or in
vials using a
Geno/Grinder.
References
Pourrut, Xavier, Marc Souris, Jonathan S Towner, Pierre E Rollin, Stuart
T Nichol, Jean-Paul Gonzalez and Eric Leroy.
2009.
Large serological survey showing cocirculation of Ebola and Marburg
viruses in Gabonese bat populations, and a high seroprevalence of both
viruses in Rousettus aegyptiacus.
BMC Infectious Diseases 2009, 9:159
Homogenizing with
Rotor
Stator Homogenizer
-
Commonly
referred to as handheld homogenizers, rotor stator homogenizers are
effective for soft tissues and small pieces of resilient tissues, such as
muscle. Samples with significant amounts of connective tissue or tendon
are more difficult to homogenize as the elastic tissues can get caught up
in the shaft of the rotor stator. This is less of a problem with softer
tissues.
The rotor stator needs to be matched with the sample volume. Product
literature suggests that samples as small as 30 ul can be homogenized,
but in practice that volume isn't practical. Generally samples of 200 ul
up to many liters can be processed, but the correct attachment is needed.
Assuming the correct rotor stator is available, place the sample in a
tube or beaker that is sufficiently narrow so that the slots on the
bottom of the rotor stator are easily submersed.
If protein degradation is an issue, the sample container can be placed on
ice turning the processing. The shaft will generate heat, thus cooling
the sample can slow down unwanted reactions.
-
Turn the
homogenizer on low and slowly work the rotor stator into the sample.
Avoid raising the rotor stator out of the buffer as air will be whipped
into the homogenate. Air and frothing can be detrimental to many
proteins.
-
The speed can
be slowly increased so that the homogenate circulates through the tip of
the rotor stator. Harder, more resilient tissues will need more
processing to breakdown fibrils.
-
Homogenization
can be optimized by measuring the release of cellular enzymes. We
commonly use lactate dehydrogenase to measure homogenization efficiency.
A time course assay can be performed on the homogenate and assayed for
enzyme release. For more on the LDH assay see this
link.
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