## 4 Discussion
### Developing and Detailing a Protocol for Isolation and Characterization of Gingipains
#### 4.1.3 Characterization of Gingipains  
SDS-PAGE  
_Sample preparation_  
1. Transfer 50 \(\mu\)L of gingipains sample into the sterile 1.5 mL microcentrifuge tube. 2. Mix the gingipain sample with 10 \(\mu\)L of 5\(\times\) non-reducing sample solubilizing buffer and 6 \(\mu\)L of 10% \(\beta\)-mercaptoethanolhanol. 3. Boil the sample for 10 min at 100 \({}^{\circ}\)C.
4. Load the samples onto SDS-polyacrylamideamide gel. _Perform Gel electrophoresis_  
Run the gels at 110 V constantly for 90 min. [Note: The timing of electrophoresis should be adjusted depending to the samples and gel size]  
_Gel staining_  
1. Stain the gel with Coomassieie gel staining solution for 30 min at room temperature while agitating it gently. 2. De-stain the gel with the de-staining solution for 30 min until the clear and sharp, blue-colored bands are visible. _Gel viewing_  
View the clear and sharp, blue-colored bands form on the SDS-polyacrylamideamide gel with UV transilluminator. Gelatin Zymography  
_Sample preparation_  
1. Transfer 5 \(\mu\)L of gingipains sample into the sterile 1.5 mL microcentrifuge tube. [Note: Sample volume varies depending on target protein]
2. Mix the gingipain sample with 1 \(\mu\)L of 5\(\times\) non-reducing sample solubilizing buffer (without \(\beta\)-mercaptoethanol)
3. Add 200 \(\mu\)M of Leueptin inhibitor (Rgp selective inhibitor), 20 \(\mu\)M of Cathepsin B inhibitor II (Kgp selective inhibitor), and 2 mM of TLCK (general inhibitor) into gingipains samples separately. [Note: In a condition to determine specific proteases, an inhibitor can be incorporated]
4. Incubate the samples for 15 min at room temperature. [Note: Do not heat the sample because it will inactivate the proteinase's enzymatic activities.] 5. Load samples onto SDS-gelatin-polyacrylamideamide gel. _Perform Gel electrophoresis_  
Run the gels at 110 V constantly for 90 min. [Note: The timing of electrophoresis should be adjusted depending to the samples and gel size]  
_Gel Renaturation/Incubation_  
1. After electrophoresis, remove the gel from the electrophoretic gel cassette carefully. 2. Place the gel in a clean plastic container and rinse it with distilled water twice for 15 min with gentle agitation. 3. Equilibrate the gel with 100 mL of renaturation buffer for 30 min at room temperature while agitating it gently. 4. Incubate and activate the proteases present in the gel with 100 mL of incubation buffer for 2 h at 37 \({}^{\circ}\)C.  
* 1. Stain the gel with Coomassieie gel staining solution for 30 min at room temperature while agitating it gently. * 2. De-stain the gel with de-staining buffer for 30 min until the transparent and sharp bands are observed. Gel viewing View the clear bands form on the SDS-gelatin-polyacrylamideamide gel with the UV transilluminator. 4.1.4. Chemicals  
Acetone (Merck KGaA, Darmstadt, Germany), Prestained protein standards (Genedi-reX, Vegas, NV, USA), Coomassieie brilliant blue R-250 (Sigma-Aldrich, USA), Acetic acid (Sigma-Aldrich, USA), Ethanol (Sigma-Aldrich, USA), Gelatin (Sigma-Aldrich, USA), Triton X-100 (Sigma-Aldrich, USA), Sodium dodecyl sulfate (Biobasic, Toronto, ON, Canada), Tris base (1 st Base, Seri Kembangan, Selangor, Malaysia) Acrylamide (Sigma-Aldrich, USA), N,N\({}^{\prime}\)-methylene bis(acrylamide) (Sigma-Aldrich, USA), Glycerol (Sigma-Aldrich, USA), Bromophenol blue (Merck, Rahway, NJ, USA), Ammonium persulfate (Bio-Rad, Hercules, CA, USA), Glycine (Bio-Rad, USA), TEMED (Bio-Rad, USA), Sodium chloride (Friendemann Schmidt, Leipzig, Germany), Calcium chloride (Nacalai Tesque, Japan), EDTA (HiMedia, Thane, India), Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (J&K Scientific, San Jose, CA, USA), Sodium azide (Sigma-Aldrich, USA), N&-Benzoy-L-arginine 4-nitroanilide hydrochloride (Sigma-Aldrich, USA), Ac-Lys-pNA hydrochloride (Bachem, Bubendorf, Switzerland), Leueptin hemisulfate (Nacalai Tesque, Japan), cathepsin B inhibitor II (Sigma-Aldrich, USA). TLCK inhibitor (Sigma-Aldrich, USA), DMSO (Sigma-Aldrich, USA). 4.1.5.