Tissue Sample Preparation for Proteomic Analysis - Proteomic and Metabolomic Approaches to Biomarker Discovery - page 42

tetra uoroethylene, minimizes heat generation

while providing a nonstick surface to minimize
sample loss. 24 It is capable of homogenizing
from 0.5 to 10 grams of tissue. This type of
homogenizer is particularly suitable for soft
tissue such as brain or liver. It is not suitable
for samples in the microgram quantity and can
handle only one sample at a time.

through the narrow gap between the rotor and
stator and forced out between the teeth of the
stator, resulting in cutting action (e.g., the Poly-
tron c, USA). 26

The working part of the equipment that contacts
the samples is called the generator. The gener-
ator is typically attached to an internal rotor
driven by a motor running at speeds between
3,000 and 27,000 rpm. Typical mechanical
grinders contain multiple generators that are
selected based on a given tissue type and size.

homogenizer, Fisher Scienti

Stainless-Steel Homogenizers/Pulverizers
A freeze-fracturing tissue pulverizer is
a typical example of a stainless-steel grinder
(e.g., BioPulverizer, Bio Spec Products Inc., OK,
USA). It rapidly fragments previously frozen
tissue samples into powder in the range of 0.1
to 10 mg. 25 Liquid nitrogen is used to make the
tissue hard and brittle. This frozen tissue is
then inserted into the precooled pulverizer and
manually crushed. The tissue is turned into ice
powder and the proteins are extracted quickly
and ef

Cryogenic Homogenizers/Grinders
Cryogenic grinding involves cooling a tissue
below its embrittlement temperature with a cryo-
genic

fluid, typically LN 2 or carbon dioxide.
Tissue freezing and fragmentation occur in
seconds and no heat is generated during
grinding, preserving proteins and other biomol-
ecules during the disruption process. Probably
the greatest bene ciently into a buffer. The disadvantage

of using pulverizers is that they must be cleaned
and dried after each use, which is time
consuming if multiple samples are processed.
However, they are affordable and fast and can
also process soft, elastic tissues.

t provided by cryogenic
grinding (e.g., Freezer/Mill , SPEX SamplePrep,
NJ, USA) is the ability to grind soft or elastic
tissues that could be ground only with long cycle
times and high energies, which can alter the
viability of proteins and other biomolecules.
Cryogenic grinding also reduces the tissue
into small particles that are dif cult or impos-

sible to obtain using common mechanical
grinders. 27

Apparatuses for Tissue Homogenization
Different apparatuses have been used for
tissue homogenization, including mechanical
grinders, cryogenic grinders, bead beaters, and
ultrasonic disruptors. Some require cleaning
after each sample

Bead-Beating-Based Homogenizers/
Disruptors
Bead beaters disrupt and homogenize
samples by violently agitating and accelerating
the beads (i.e., grinding media) against the tissue
(BeadBeater , Biospec). This cyclic motion (i.e.,
2,500 to 5,000 rpm) generates strong shear forces
that disrupt tissues and release intracellular
biomolecules into the extraction buffer. 28 This
equipment is durable and simple to operate.
Initially, these devices were capable of homoge-
nizing one sample at the time ( Figure 2 A).
However, recent developments in bead-beating
technology have produced mill homogenizers

s processing; others use dispos-
able vials or tubes, which are preferred for pre-
venting cross-contamination between samples.

'

Mechanical Rotor/Stator e Type
Homogenizers/Grinders
Mechanical rotor and stator type of grinders
(i.e., handheld, bench top) consist of a rapidly
spinning propeller positioned within an open-
ended tube with holes near the working end.
Using the mechanical grinder, the sample is
vigorously mixed, accelerated, and pressed