Important parameters in semi-dry electrophoretic transfer.
The efficiency of semi-dry electrophoretic transfer after sodium dodecyl sulfate (SDS)-electrophoresis using PhastGel media was investigated in a model system using three isotope labelled proteins. To give a full picture of the blotting process the amount of protein present in the gel, membranes, and filter papers was determined after different transfer times. The influence of the transfer buffer, commonly used additives such as methanol and SDS, and several different immobilizing matrices was investigated. Soybean trypsin inhibitor, bovine serum albumin, and ferritin were used as model proteins to study the effect of size on transfer efficiency. Basically, all three stages of the blotting process decide the result; the elution of protein from the gel, the immobilization of protein to the membrane, and the loss of material from the membrane during transfer. A theoretical explanation for the observed poor binding to a second membrane is discussed. Our results show that the buffer composition has little influence on the efficiency of transfer from the gel, but can be significant to the binding capacity of the membrane. In all experiments performed, there was never one moment during the transfer when all protein was eluted from the gel and simultaneously still bound to the membrane. The highest recovery in the membrane was obtained at different time intervals for different proteins. This indicates that quantitative transfer procedures cannot be generalized. However, obtaining an optimal method for reliable quantification of a specific protein or group of proteins is possible. For general protein staining of nitrocellulose and polyvinylidene difluoride membranes, a highly sensitive silver staining method requiring only 15 min has been used.
Pharmacia LKB Biotechnology AB, Uppsala, Sweden.
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Kårsnäs
D002021:Buffers; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D005293:Ferritins / Q000032:analysis; D005782:Gels; D008567:Membranes, Artificial; D009757:Nylons; D011145:Polyvinyls; D011506:Proteins / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000032:analysis; D012834:Silver; D012967:Sodium Dodecyl Sulfate; D013194:Staining and Labeling; D014361:Trypsin Inhibitors / Q000032:analysis; D005465:Fluorocarbon Polymers
D002021:Buffers; D003101:Collodion; D005293:Ferritins; D005782:Gels; D008567:Membranes, Artificial; D009757:Nylons; D011145:Polyvinyls; D011506:Proteins; D012710:Serum Albumin, Bovine; D012834:Silver; D012967:Sodium Dodecyl Sulfate; D014361:Trypsin Inhibitors; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers