**2.3.1.2 PAGE alkaline proteins (+), Reisfeld et al. (1962)**


Methyl green Glycerol

Sample preparation:

 In a sterile microtube put 18 μL of sample and 2 μL of running buffer consisting of 31.2 g of β-alanine, 8 mL of glacial acetic acid and an amount of distilled water sufficient to reach the volume of 1000 mL;


Add 2 μL of the solution Methyl green 0.1% and 4 µL of glycerol.

Table 1. Preparation of 8.4 mL of PAGE in different concentrations.


In a sterile microtube, put 18 μL of proteic sample and 2 μL of running buffer Tris-HCl

KOH (1 M) 48 mL

Glacial acetic acid 17.2 mL TEMED 4 mL Complete with distilled H2O 100 mL

Acrylamide 19.2 g Bis-acrylamide 0.54 g Complete with distilled H2O 40 mL

ammonium persulfate 0.28 g Complete with distilled H2O 100 mL

 In a sterile microtube put 18 μL of sample and 2 μL of running buffer consisting of 31.2 g of β-alanine, 8 mL of glacial acetic acid and an amount of distilled water sufficient to

Solution Polyacrylamide concentration (%)

A (mL) 1.05 1.05 1.05 1.05 1.05 1.05 1.05 1.05 C (mL) 0.71 0.876 1.05 1.22 1.49 1.77 2.10 2.45 H2O (mL) 6.65 6.47 6.30 6.13 5.97 5.60 5.25 4.95

4 5 6 7 8 10 12 14

Add 2 μL of the dying solution Bromophenol blue 0.1% and 4 µL of glycerol.

Preparation of the samples and markers:

50mM and glycine 36mM, pH 8.9;

Sample preparation:

reach the volume of 1000 mL;

Set up the electrophoresis bowl;

**2.3.1.2 PAGE alkaline proteins (+), Reisfeld et al. (1962)** 

**Solution A** 

**Solution C** 

**Solution G** 

Methyl green Glycerol

Add 2 μL of the solution Methyl green 0.1% and 4 µL of glycerol.

Table 1. Preparation of 8.4 mL of PAGE in different concentrations.

In a Becker, add the solutions A, C and H2O. Mix;

**Dye** 

pH adjusted for 4.3

pH adjusted for 4.5


#### **2.3.2 Electrophoresis gel SDS-PAGE, Laemmli (1970)**

The electrophoresis must be performed in pH 8.9 and in the presence of SDS (sodium dodecyl sulfate), with the gel concentration ranging from 5 to 15%. Table 2 indicates necessary volumes to obtain SDS-PAGE gels with different concentrations.


Gel Electrophoresis for Investigating Enzymes with Biotechnological Application 105

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.,

Method I – After conducting a PAGE 4.5 or 8.9 (depending on the enzyme pI), the gel containing the enzyme must be incubated with the substrate – a solution containing citric pectin or sodium polypectate 1% in the suitable buffer of the enzyme under study. In Fig. 2B there was the use of 1% of sodium polypectate in a sodium acetate buffer 100mM, pH 4.0 and incubation at 50ºC (enzyme optimum temperature), for 2 hours for the dying with 0.02% Ruthenium red [(Ru3O2(NH3) 14)Cl6.4H2O)], a dye capable of interacting with the pectic substates (Sterling, 1970). Thus, in the region where the protein migrated to and hydrolyzed the substrate, there is a halo with a whitened coloration that contrasts against

Method II – The citric pectin must be dissolved in gel buffer with the aid of a magnetic agitator, followed by the addition of acrylamide, bis-acrylamide and TEMED solutions. Crystals of ammonium persulfate are added immediately before the plate gel is overflown. After the run, incubate the gel for 1-2 hours with 100 mL of malic acid 0.1M, at 4ºC, in order to cause a gradual change to pH 3.0. Such period allows the enzyme to interact with the pectin polymerized in the acrylamide gel in its suitable pH range. Wash with distilled water

Result: against a redish gel, it is possible to notice the polygalacturonase activity due to the

In order to detect the xylanase activity in gel, the polyacrylamide must be polymerized with 0.5% xylan dissolved in the buffer of the electrophoresis to be performed (PAGE 4.5 or 8.9, depending on the isoelectric point of the enzyme under study). After the electrophoresis run the gel must be incubated in the temperature and in the reaction buffer which is mostly suitable for the xylanase under study for at least 1 hour. After this period, the gel is going to be stained with 1% Congo red (C32H22N6Na2O6S2) a sodium salt of benzidinediazo-bis-1 naphtylamine-4-sulfonic acid. Thus, in the region where the protein migrated to and hydrolyzed the substrate, there is a halo with a whitened coloration that contrasts against

Xylanases may also be observed through zymograms (Fig. 2D). For so, the samples must be applied in SDS-PAGE without the addition of β-mercaptoethanol, any other reducing agent

and color in Ruthenium red 0.02%, during 30 to 120 min. Wash with distilled water.

the rest of the red-colored gel. (Fig. 2C) (Sandrim et al., 2005; Damásio et al., 2011).

**3. Methods for specific enzymes detection in electrophoresis gel** 

**3.1 Amylase** 

**3.2 Pectinase** 

**3.3 Xylanase** 

2001; 2003; Silva et al., 2009a, 2009b).

the rest of the red-colored gel.

formation of clear, opaque or colorless areas.

Preparation of samples and markers:



Table 2. Preparation of SDS-PAGE


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) development of the electrophoresis.
