Protein Analysis - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 166

4. [Optional] Subject the sample to sonication (fi ve consecutive
short 5 s pulse with 10 s interval). The sample must be dipped
in ice during each interval to remove the heat generated during
sonication. (Please optimize based on the types of sonication
system used.)
5. The sample is then subjected to a high-speed centrifugation
(14,000 × g , 30 min, 4°C). The resulting supernatant with the
pellet removed will be the fi nal state of the sample ready for the
following assays.

In this chapter, we have presented methods to prepare total cell
lysate from tissues and cell lines. Nevertheless, in cases where iden-
tifi cation of transcription factors that bind to specifi c DNA or RNA
sequences, purifi ed organelle-specifi c protein fractions such as
nuclear or mitochondrial protein fractions can be used in lieu of
total cellular proteins to EMSA or chIP assays to enhance the
chances of success. Preparation of such specifi c organelle proteins
are beyond the scope of this chapter.

2.3. Variations

3. Western Blot
Analysis

Western blot analysis is a common technique used to identify and
compare quantitative changes of specifi c proteins. It is comprised
of two parts: (1) resolving the pool of proteins within the sample
followed by transferring the protein profi le onto a membrane and
(2) identifying the protein of interest using specifi c antibody rec-
ognizing it. Western blot analysis is semiquantitative, quantitative
changes of the identifi ed protein between samples are refl ected by
the change in intensity of the band representing the protein.
However, there is a linear range limit when quantifying via Western
blot analysis. Hence tittering of the total protein of the samples is
a good practice to ensure the signal is within linear range limit.
In this chapter, we present the procedure for SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) which is the most common tech-
nique used in all research studies.

3.1. Material
and Instruments

Part 1

1. 1.5 M Tris (pH 8.8) solution.
2. 1 M Tris (pH 6.8) solution.
3. 30% Acrylamide/Bis Solution, 29:1 (Bio-rad).
4. 10% w/w Ammonium persulfate solution (APS).
5. N , N , N ¢, N ¢-Tetramethylethylenediamine (TEMED) (Sigma).
6. 10% SDS solution.
7. 4× Sample Buffer (60 mM Tris-Cl, 25% glycerol, 2% SDS,
14.4 mM 2-mercaptoethanol, 0.1% bromophenol blue).

3.1.1. Materials