western blot protocols, troubleshooting, scientific resources and research atricles for the research community and science students
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In order to be able to use a western blot to study a given ion channel, proteins need to be ‘accessible’. This is achieved by performing a protein extraction. Several extraction methods are available and the approach one takes will depend on the specific protein characteristics (e.g., cellular localization). Since ion channels are membrane proteins, in order to obtain the highest concentration of relevant proteins during sample preparation, a membrane protein extraction is performed. This protocol differs from total protein extraction by concentrating membrane proteins in a smaller volume of buffer. Given the sample volume loading constraints of a typical polyacrylamide gel, if the ion channel that is being studied has a low copy number, it is important to have a high membrane protein extraction yield.
The appropriate use of protease and phosphatase inhibitors should help preserve subunit proteins during extraction, however some ion channel subunits are more sensitive to degradation than others. There may be circumstances where the additional processing time involved with membrane protein extraction warrants the use of the more straightforward total protein extraction.
In cases where
the channel subunit copy number is too low to obtain a good quality western
blot, microcentrifuge filtration units can be used to concentrate the
samples. These units consist of two parts:
1) a loading cup with filter (filters are made of various materials and
come in several pore sizes) and
2) a microcentrifuge tube for filtrate collection.
The concept is traighforward: load the protein extracts in the cup and
use centrifugation to filter the sample. Proteins larger than the filter
pore size will stay in the top chamber, the rest will collect in the bottom,
along with the sample buffer.
Once the protein sample has been retrieved and resuspended in sample buffer, analysis by western blot protocol is possible.
1. PubMed: comprehensive database - 15 million citations
2. NIH:the National Institutes of Health
3. UniProt: The world's most comprehensive catalogue of information on proteins
4. Western
Blot Procedure Western blot protocol overview.