Protein Analysis - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 171

Fig. 4. The bands on the X-ray fi lm indicating specifi c protein of interest.

is to be incubated overnight, then it should be at 4°C to
prevent bacteria, fungi, and mold contamination.
3. Then, the membrane is washed for 5 min in TBST consecu-
tively for at least three times.
4. After the washes, the membrane is to be incubated with the
appropriate secondary antibody (one that recognizes the
specifi c species of the primary antibody). The optimal dilution
of the secondary antibody is also dependent on the source of
the antibody. Typically, 1-h incubation at room temperature
would suffi ce.
5. Again, the membrane is washed for 5 min in TBST consecu-
tively for at least three times.
6. After the washes, the membrane is then subjected to a chemi-
luminiscent system enabling the HRP conjugated onto the
secondary antibody to react with its substrate to generate
chemiluminiscence. The actual procedure may differ based on
the commercial source of the system.
7. The chemiluminiscence is detected by exposure to X-ray fi lm
in the dark room. The protein of interest is indicated as bands
on the developed fi lm as follows with the intensity refl ecting its
quantity in the sample as shown in Fig. 4 .

What is outlined above is called the SDS-PAGE where all the pro-
teins are denatured and surrounded by SDS. Hence, the resolution
of the proteins is based solely on the size of the proteins. Nevertheless,
a variation of this is the native PAGE where SDS is removed from
the PAGE, the Running Buffer as well as the Sample Buffer. In cases

3.3. Variations