Calcium ion improves electrophoretic transfer of calmodulin and other small proteins.
Inclusion of 2 mM CaCl2 in standard electrotransfer buffer improves detection of transferred calmodulin 20-fold. Detection of the calcium-binding proteins calmodulin and alpha-lactalbumin displays similar improvement, some other small proteins show slightly improved detection, while other proteins, especially those greater than or equal to 30 kDa, show none. The effect of CaCl2 on transfer is a result of enhanced retention of protein by the binding matrix. Our results suggest that addition of CaCl2 more effectively removes sodium dodecyl sulfate from proteins, increasing protein-binding to polyvinylidene difluoride and nitrocellulose membranes. The enhanced membrane-binding of calmodulin is not due to specific binding of calcium ion, as other divalent cations are similarly effective. Detection of native calmodulin is also slightly enhanced after transfer with added CaCl2, suggesting an additional, minor effect of the added ion. The increased binding brought about by inclusion of calcium ion in transfer buffer offers a very useful means for improving the sensitivity of assays that involve electro-transfer.
McKeon
T A
TA
Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710.
Lyman
M L
ML
D000818:Animals; D002021:Buffers; D002118:Calcium / Q000737:chemistry*; D002147:Calmodulin / Q000032:analysis*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008567:Membranes, Artificial; D011506:Proteins / Q000032:analysis
D002021:Buffers; D002147:Calmodulin; D008567:Membranes, Artificial; D011506:Proteins; D002118:Calcium