**3.3 Xylanase**

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

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

Gel Electrophoresis for Investigating Enzymes with Biotechnological Application 107

removed. For so, add 70 mL of buffer with the appropriate reaction pH, incubate for 30 min.,

**A B C D E**

Fig. 2. Activity gels for different enzymes in SDS-PAGE 10%. (A1) α-glucosidase (a type of amylase) revealed with Coomassie Blue; (A2) α-glucosidase revealed with 10 mM Iodine solution and 14 mM potassium iodide; (B1) polygalacturonase revealed with silver solution; (B2) polygalacturonase activity revealed with 0.02% ruthenium red; (C1) xylanase revealed with silver solution; (C2) xylanase activity revealed with Congo red; (D1) Zymogram for xylanase revealed with silver solution and (D2) Congo red; (E) protease activity revealed

Following, remove the buffer and add the casein solution at 3%, prepared in a buffer with the enzyme reaction pH. The gel must be incubated for 30 min, at 4ºC, for the diffusion of the casein to the gel. After that, the gel must be bathed at the enzyme reaction temperature for the period of 1-2 hours, so that the enzymatic reaction occurs (such period may be

The excess casein must be removed by bathing the gel for 5 times with distilled water at room temperature (García-Carreño et al., 1993 and Kleiner & Stetler-Steveson, 1994) with

For the coloration, the gel must be stained with a solution containing 40% ethanol, 10% acetic acid and 0.1% Coomassie brilliant blue R-250 (García-Carreño et al., 1993). In this stage, the gel shows a blue bottom and where the hydrolysis took place, there will be a white halo. The gel needs to have the excess Coomassie brilliant blue removed; hence, it will have to be discolored with a discoloring solution composed by 40% ethanol and 10% acetic acid (García-Carreño et al., 1993). The clear zones over the blue bottom indicate the protease

Some enzymes need activators, such as Ca2+, DTT and EDTA to show their activity. Those can be added together with the substrate. Other substrates, such as hemoglobin, bovine serum albumin, gelatin and collagen, may have their coloration improved through the use

of other dyes, as for example, Amide black (García-Carreño et al., 1993).

**1 2 12 12 12**

at 4°C, 100rpm.

with Coomassie Blue.

modifications.

activity.

adjusted according to each enzyme and concentration).

and without boiling. The advantage of the activity detection through zymograms is that effectively all active isoforms present there may be detected, once that regardless on the isoelectric point, all proteins will migrate in the gel, because of their molar mass.

In this case, the SDS gel must be performed with the running buffer commonly adopted in the protocols and, after the electrophoresis run, the proteins will have to be transferred to another gel composed by agarose and xylan, a substrate that is specific to the activity to be detected. Such transferring happens when there is a kind of "sandwich" with the polyacrylamide gel and the agarose + substrate gel. The transferring must happen overnight. After this period, the agarose + substrate gel, now also with the proteins to be analyzed, will have to be incubated in the buffer that is suitable for the isoforms under study, during one hour, following with the coloration suitable for the activity detection of the enzyme analyzed.
