Conditions for improved adsorption of calmodulin to nitrocellulose: detection by 45Ca binding.
The 45Ca filtration method which allows calcium-binding proteins to be detected after their adsorption to nitrocellulose filters was used to test conditions for improved Western blotting of calmodulin. Our results indicate that dodecyl sulphate is not efficiently removed from calmodulin during electroelution in standard transfer buffers. This detergent disrupts binding of calmodulin to nitrocellulose. Electrotransfer in potassium buffer which precipitates dodecyl sulphate leads to a 6-fold increase in calmodulin retention by nitrocellulose. With this and other modifications, the detection limit of the 45Ca overlay method of Maruyama et al. (J. Biochem. 1984, 95, 511-519) for calmodulin is increased to 0.2 microgram per lane.
Hincke
M T
MT
Department of Anatomy, University of Ottawa Faculty of Health Sciences, Canada.
D000327:Adsorption; D015153:Blotting, Western; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D003101:Collodion; D000431:Ethanol / Q000494:pharmacology; D005374:Filtration; D010710:Phosphates / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D017680:Potassium Compounds; D012967:Sodium Dodecyl Sulfate
D002021:Buffers; D002147:Calmodulin; D010710:Phosphates; D017680:Potassium Compounds; D012967:Sodium Dodecyl Sulfate; D000431:Ethanol; D003101:Collodion; C013216:potassium phosphate; D011188:Potassium; D002118:Calcium