Protein Analysis - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 169

30%
Acrylamide
mix

1.0 M Tris
(pH 6.8) 10% SDS H 2 O 10% APS TEMED

Stacking
gel

0.67 ml 0.55 ml 50 ml 2.7 ml 30 ml 3 ml

6. Immediately after the stacking gel mixture is pour on top of
the resolving gel, the sample comb is inserted into the stacking
gel mixture to form sample wells as shown in Fig. 2 .
7. The stacking gel is allowed to polymerize for 15 min.
8. Meanwhile, samples of equal total amount of the proteins are
normalized to the same concentration by adding Lysis Buffer.
Then, each sample is added with an appropriate amount of 4×
Sample Buffer before heating it to 95°C. This will enhance
protein denaturation and SDS to surround all proteins.
Afterward, the samples are ready for loading onto the gel.
9. Once the stacking gel is set, the comb is removed giving rise to
wells where the samples are to be loaded. One of the well is
reserve for prestained markers to enable the estimation of
protein sizes.
10. Once the samples and prestained markers are loaded, protein
resolution begins as soon as the Protean system is connected
to the power supply and a voltage of 80-100 V is applied.

Fig. 2. Casting of a polyacrylamide gel with the comb defi ning the sample lanes.