3.1.1 Membrane Selection

Membranes (for Western Blotting)
A membrane generally refers to a porous material with a pore size of 0.1–0.45 μm, and its binding capacity mainly depends on the pore size.
Membranes with many small pores have a larger binding surface area than those with larger pores, so they usually have higher binding capacity.
The exact mechanism of interaction between biomolecules and membranes remains unclear; it is currently widely believed to be a combination of non-covalent forces and hydrophobic forces.
Although protein conformation and buffer composition also affect binding capacity, the sensitivity of Western blotting mainly depends on the total amount of protein immobilized on the membrane that can bind to the primary antibody.
However, excessive protein binding does not necessarily increase the signal intensity of Western blotting (WB); instead, it may have the opposite effect.
This is because high-concentration proteins bind to each other through weak interactions, and this binding force is stronger than the binding force between proteins and the membrane.
This means that protein/antibody complexes can be easily washed off the membrane during the washing step.
Experimental Procedures
1. Membrane Selection Guide
Molecular Weight of Target Protein Membrane Pore Size
≤ 10 kDa 0.1 μm
≤ 20 kDa 0.2 μm
≥ 20 kDa 0.45 μm
2. Nitrocellulose (NC) and PVDF Membranes – Comparison of Properties and Applications
NC and PVDF membranes are the most commonly used membranes for Western blotting. Their properties and applications are compared as follows:
Property Nitrocellulose Membrane (NC) PVDF Membrane
Protein Binding Capacity 80–100 μg/cm² 100–300 μg/cm²
Total Protein Staining Methods Colloidal gold, Ponceau S, Amido Black, India ink, Sypro blot dye Colloidal gold, Ponceau S, Amido Black, India ink, Coomassie Brilliant Blue
Detection Methods Chromogenic, chemiluminescence, fluorescence, radioactivity Chromogenic, chemiluminescence, fluorescence, chemifluorescence, radioactivity
Indirect Blotting Method No Yes
Rapid Immunoassay No Yes
Western Rehybridization No Yes
Edman Sequencing No Yes
Amino Acid Analysis Yes Yes
Protein Binding in Presence of SDS Poor Good
On-Membrane Digestion/Mass Spectrometry No Yes
3. Formulation of Transfer Buffer
10× Transfer Buffer (1 L):
Weigh 30.3 g of Tris and 144 g of Glycine.
Add approximately 800 mL of deionized water, and stir to dissolve completely.
Adjust the volume to 1 L with deionized water.
Store at room temperature.