Biochemical and Molecular Biological Assessments of Intracerebral Hemorrhage - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 636

4. The smaller the volume loaded onto the gel, the better the
resolution will be.
5. The proteins should be run slowly through the resolving gel to
prevent “smiling” of the separated polypeptides, where the
bands toward the edges of the gel tend to turn upward.
6. Good contact between the gel and the nitrocellulose is essential
for effi cient electrotransfer, and care should be taken to ensure
that all air bubbles are removed, as these will prevent uniform
current fl ow, and thus interfere with the blotting process.
7. The process of “blocking” the membrane after transfer is essen-
tial to block the unoccupied protein-binding sites on the mem-
brane. Failure to carry out this step effectively will result in the
primary and secondary antibodies binding to these sites, lead-
ing to a reduction in the sensitivity of the blotting process and
unacceptably high background “Noise.” The presence of 0.1%
(v/v) of polyethylene sorbitan monolaurate (Tween-20) in all
buffers during the processing of the membrane assists in the
prevention of nonspecifi c binding.

2. Enzyme-Linked
Immunosorbent
Assay

Enzyme-linked immunosorbent assays (ELISAs) combine the
specifi city of antibodies with the sensitivity of simple enzyme assays,
and are used to detect the presence and concentration of an anti-
gen or an antibody in a sample. There are many different types
of ELISAs. One of the most common types of ELISA is the
“sandwich ELISA.”

2.1. Sandwich ELISA

The direct sandwich ELISA involves the passive attachment of anti-
body to the solid phase. The antibody (capture antibody) then bind
antigen that is added. The antigen is diluted in a blocking buffer to
avoid nonspecifi c attachment to the solid phase. After incubation
and washing, an antibody-antigen complex is attached to the solid
phase. The captured antigen is then detected by the addition and
incubation of enzyme-labeled specifi c antibody in blocking buffer.
Thus, this is a direct conjugate binding with the antigenic targets
on the captured antigen. After incubation and washing, the bound
enzyme is developed by the addition of substrate/chromogen, then
stopped, and fi nally read using a spectrophotometer.

2.2. TNF- a
Measurement

Here, we provide an ELISA for quantitative determination of
TNF-a in brain tissue ( 5 ).

Following ICH induction, basal ganglia samples are prepared.
The tissues are diluted (40% weight/volume) with 0.01 mol/L

2.2.1. Sample Preparation