Protein Analysis - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 167

8. Running Buffer (25 mM Tris-Cl, 192 mM glycine, 0.1% SDS).
9. Transfer Buffer (15.6 mM Tris-Cl, 120 mM glycine, 20% v/v
methanol).
10. PVDF or nitrocellulose membrane.
11. Blotting paper.

Part 2

1. TBST (TBS with 0.5% Tween 20)
2. Blocking Buffer (5% nonfat milk solution in TBST)
3. Primary Antibody
4. Secondary Antibody conjugated with HRP (horse radish
peroxidase)
5. Chemiluminiscent system
6. X-ray fi lm

Part 1 3.1.2. Instruments

1. Mini-Gel Apparatus: We are going to use the Bio-Rad Protean
II system as a model since it is the most commonly used system
to date. Nevertheless, all systems have very similar makeup.
2. Power supply (capacity 200 V, 500 mA).
3. Heat block that can heat up to 100°C.

Part 2

1. Electro-transfer Apparatus: We are going to use the Bio-Rad
protein transfer system as a model. This system shares some of
the same hardware with the Protean II system.
2. Film developer.

3.2. Procedures

Part 1
1. To cast a mini-gel, we fi rst set up the casting apparatus in
accordance to the manufacturer's instruction. The setup
apparatus is shown in Fig. 1 .
2. The percentage of the resolving gel needed is dependent on
the size of the proteins of interest. The following table provides
some guidance to the most suitable percentage gel for your
needs.

Acrylamide percentage (%) Size of proteins (kDa)

15 15-45

12 15-65

10 20-75

8 30-120