Experimental Procedures

1. Sample Preparation

1.1 Sample Types

• 1.1.1 Conventional Mammalian Adherent Cells
• 1.1.2 Mammalian Suspension Cells
• 1.1.3 Mammalian Soft Tissues
• 1.1.4 Animal Blood Proteins
• 1.1.5 Bone Tissues
• 1.1.6 Bacteria
• 1.1.7 Yeast
• 1.1.8 Insect Cells
• 1.1.9 Plant Tissues
• 1.1.10 Membrane Proteins
• 1.1.11 Organelles/Suborganelles
• 1.1.12 Nuclear/Cytoplasmic Protein Extraction
• 1.1.13 Mitochondrial/Cytoplasmic Fraction Extraction
• 1.1.14 Endoplasmic Reticulum Extraction
• 1.1.15 Exosome Extraction
• 1.1.16 Protein Extraction from FFPE Samples

1.2 Protein Quantification

• 1.2.1 UV Spectrophotometry
• 1.2.2 BCA Assay
• 1.2.3 Coomassie Brilliant Blue Assay

1.3 Protein Denaturation

2. Sample Loading and Electrophoresis

2.1 Sample Loading

• 2.1.1 Sample Loading
• 2.1.2 Protein Ladder (Marker)

2.2 Electrophoresis

• 2.2.1 Denaturing Gel SDS-PAGE
• 2.2.2 Gel Preparation
• 2.2.3 Native Polyacrylamide Gel Electrophoresis (Native-PAGE)
• 2.2.4 Tris-Acetate Gel

3. Membrane Transfer

3.1 Products Required for Membrane Transfer

• 3.1.1 Membrane Selection

3.2 Procedures

• 3.2.1 Membrane Transfer Methods

3.3 Total Protein Staining

3.4 Experimental Tips

4. Blocking

4.1 Reagents Required for Blocking

4.2 Experimental Tips

5. Antibody Incubation/Detection

5.1 Formulations of Reagents to Be Prepared for Antibody Incubation

5.2 Procedures

• 5.2.1 Antibody Incubation
• 5.2.2 Detection
• 5.2.3 Sequential Incubation of Additional Targets on the Same Membrane

5.3 Experimental Tips

6. Troubleshooting

• 6.1 No Signal
• 6.2 High Background & Multiple Bands
• 6.3 Incorrect Band Size
• 6.4 Others

7. Experimental Tips for Common WB Targets

• 7.1 Caspase 3
• 7.2 LC3B
• 7.3 NF-κB
• 7.4 P-AKT
• 7.5 NLRP3
• 7.6 E-Cadherin
• 7.7 p53