How to Perform Western Blot for High Molecular Weight (500 kDa) Proteins
When analyzing high molecular weight proteins (e.g., 500 kDa) using Western blotting, it is often necessary to optimize specific parameters to ensure effective resolution and detection. The following aspects should be considered:
1. Gel Selection
Low-percentage polyacrylamide gels (e.g., 4–8%) are more suitable for resolving high molecular weight proteins.
2. Electrophoresis Conditions
Running the gel at a low voltage helps minimize protein overheating and denaturation while allowing sufficient time for the migration of large proteins through the gel matrix.
3. Transfer Conditions
The wet transfer method is generally more effective than semi-dry transfer for large proteins. Prolonging the transfer duration and/or increasing the current can enhance transfer efficiency and ensure complete protein migration onto the membrane.
4. Membrane Selection
Polyvinylidene difluoride (PVDF) membranes are typically more effective than nitrocellulose membranes for the transfer of high molecular weight proteins.
5. Blocking and Antibody Incubation Conditions
Adequate blocking, incubation, and washing conditions—both in duration and temperature—are critical for reducing background noise and enhancing the specificity of antibody binding.
6. Molecular Weight Markers
Employ molecular weight markers that include high molecular weight standards to accurately assess the migration and molecular weight of the target protein.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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