**3.1 Amylase**

104 Gel Electrophoresis – Advanced Techniques

 In a sterile microtube, place a sample buffer and a molar weight marker in the ration of 1:1; In a sterile microtube, place the buffer sample and the sample in the ratio of 1:3.

Add the ammonium persulfate and quickly place the solution in the electrophoresis

Wait until the gel solidifies and apply the patterns (0.5-5 μL) and the samples (up to 30

Connect the energy cables in the respective jacks of the source regulated for 70 mAmps

**Mini Tank**

**Sample Run Buffer**

**Electrophoresis Power Supply**

**Lid**

**Electrode Assembly**

**Banana Plug Jacks Notch on U-Shaped Gasket**

Connect the source and wait until the samples run throughout the gel before turning it off;

**A B**

**C D**

**E F**

**G**

Fig. 1. Preparation and development of vertical polyacrylamide electrophoresis gel using Bio-RadTM system. (A) and (B) accessories for stacking gel. (C) and (D) preparation of the

electrophoresis gel; (E) addition of run buffer; (F) application of the samples; (G)

 5 6 7 8 10 12 A (mL) 1 1 1 1 1 1 C (mL) 1.4 1.7 2 2.3 2.85 3.45 E (mL) 3.8 3.8 3.8 3.8 3.8 3.8 H2O (mL) 1.8 1.5 1.2 0.9 0.35 - Ammonium persulfate(g) 0.00425 0.00425 0.0057 0.006 0.007 0.00708

Solution/Reagent Gel concentration (%)

In a Becker, add the solutions A, C, E and H2O and mix;

Preparation of samples and markers:

Table 2. Preparation of SDS-PAGE

Set up the electrophoresis bowl;

Remove the gel carefully and process it.

**Comb**

**Run Buffer**

development of the electrophoresis.

**Gel Cassete**

**Glass Cassete Pressure Cams**

bowl; Place the comb;

μL) in different lanes;

and 120 Volts;

The method consists of the conduction of an electrophoresis in polyacrylamide gel polymerized with starch 0.5%. The electrophoretic run is performed in the pH adequated to the amylase isoelectric point. After the end of the run, the gel must be immersed in the suitable temperature and buffer during at least one hour. The gel is going to be revealed with a solution of iodine (I2 10 mM) and potassium iodide (KI 14 mM) until the appearance of activity bands. Fig. 2A illustrates the activity of α-glucosidase, one of the enzymes of the amylolytic system, which leads to the formation of glucose as end product (Aquino et al., 2001; 2003; Silva et al., 2009a, 2009b).
