A method to increase efficiency and minimize anomalous electrophoretic transfer in protein blotting.
We find that in protein blotting, the electrophoretic transfer efficiency of a given protein can be markedly affected by other components in the sample. Using a variety of electrophoretic elution protocols, we obtained very inefficient transfer of purified 125I-labeled 275-kDa cation-independent mannose 6-phosphate receptor from a sodium dodecyl sulfate-polyacrylamide gel to a membrane. Surprisingly, the transfer efficiency increased significantly if other proteins that migrated in the vicinity of the receptor were present. This may be a generally unappreciated but potentially widespread phenomenon as other but not all radiolabeled proteins examined behaved similarly. This carrier effect occurs under conditions typically used in protein blotting experiments and can lead to large errors in quantitative analysis. We developed two methods to minimize the carrier effect. First, addition of excess carrier protein to samples prior to electrophoresis saturated the carrier effect so that different samples had similar transfer efficiencies. Second, addition of either bovine serum albumin or sodium deoxycholate to the cathodic electro-elution buffer markedly increased transfer efficiency of radiolabeled proteins that exhibited poor transfer efficiency and minimized transfer differences between samples that contained or lacked carrier. These methods should be generally applicable to standard protein blotting and analysis protocols.
Bhavsar
J H
JH
Rutgers University, New Brunswick, New Jersey 08903.
Remmler
J
J
Lobel
P
P
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D015151:Immunoblotting / Q000379:methods; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D007553:Isotope Labeling / Q000379:methods; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D017527:Receptor, IGF Type 2 / Q000032:analysis / Q000302:isolation & purification*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000302:isolation & purification
D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D011506:Proteins; D017527:Receptor, IGF Type 2; D012710:Serum Albumin, Bovine