Protein Analysis - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 165

With brain or spinal cord tissues, they can be fl ash frozen in liquid
nitrogen followed by grinding them into powder in a prechilled
(liquid nitrogen) mortar and pestle before the proteins are
extracted. Alternatively, tissues can also be homogenized with a
dounce homogenizer before the proteins are extracted.

2.1. Materials
and Instruments

1. Washing Buffer TBS: 20 mM Tris-Cl (pH 7.5), 150 mM
NaCl.
2. Lysis Buffer: 0.1% Triton X-100, 20 mM Tris-Cl (pH 7.5),
150 mM NaCl, protease inhibitor cocktails*.
*Protease inhibitor cocktails are mixture of protease inhibitors
to inhibit the function of intracellular proteases and are com-
mercially available.

2.1.1. Material

1. Stainless steel mortar and pestle or
2. Glass dounce homogenizer

2.1.2. Instruments

2.2. Procedure

For brain or spinal cord tissues,

1. The tissue can be fl ash frozen in liquid nitrogen and grind into
powder in a prechilled (liquid nitrogen) mortar and pestle.
2. The powder is collected in a plastic centrifuge tube and resus-
pended in Lysis Buffer resulting in the approximate concen-
tration of 10 7 -10 8 cells/ml followed by a 15-min incubation
on ice.
3. Alternatively, the tissue (small sample) can also be placed in a
suitable glass dounce homogenizer in the presence of Lysis
Buffer prechilled on ice.
4. After 15-20 passes, carefully observed that the homogenate is
even without chucks of tissues, it is removed from the dounce
homogenizer into a plastic centrifuge tube on ice.
5. Using either method, the samples are ready to be subjected to
an optional pulse-sonication step (see step 4 for cell lines).
6. Then, the samples are subjected to a 30-min centrifugation
(14,000 × g , 4°C) and after removing the pellet, the superna-
tant is the sample ready for assays.

For cell lines,

1. Wash the cells gently once with TBS at 4°C.
2. For adherent cells, they are trypsinized to detach from the
culture plate and centrifuged (1,200 rpm, 3 min) to form a cell
pellet with the supernatant removed.
3. Resuspend the cell pellet in Lysis Buffer resulting in the
approximate concentration of 10 7 -10 8 cells/ml and incubate
on ice for 15 min.