2), the methodology of this application has changed High-molecular-weight proteins often blot onto nilittle since it was originally introduced for use with trocellulose membranes poorly following sodium dourea-or SDS 2 -containing polyacrylamide gels (1). Nudecyl sulfate-polyacrylamide gel electrophoresis, remerous attempts to improve the original protocol have sulting in low levels of detection on immunoblots, and focused on increasing the amount of protein transhence difficulty in analyzing rare proteins. Moreover, ferred to and retained on the nitrocellulose sheet, paroptimizing conditions for the transfer of high-molecuticularly when dealing with either very small or very lar-weight proteins to nitrocellulose frequently results large proteins. The observation that small proteins in the inefficient transfer or loss of lower molecular tend to move through the nitrocellulose, with only a weight proteins. This problem is particularly vexing fraction of the total amount actually binding, led to an during the analysis of large proteins which may be analysis showing that nitrocellulose with smaller pore processed to one or more smaller biologically active sizes was more effective in retaining small proteins (3). forms. Using radiolabeled protein standards and phos-More recently, Too et al. describe the use of gelatinphorimaging technology, we have quantitated the efcoated nitrocellulose for the quantitative retention of ficacy of many different protein gel electrophoresis small proteins (4). Conversely, investigators analyzing and blotting protocols. Here we report novel gel and large proteins have often sought to increase the effiblotting conditions which significantly improve the ciency of transfer by enhancing the degree of protein transfer and retention of high-molecular-weight promigration out of the gel during the transfer, using techteins, without sacrificing the efficient transfer of lower niques ranging from disruption of the gel matrix to molecular weight proteins. Using this newly described partial proteolytic digestion of the proteins prior to procedure, we have detected a rare 500-kDa protein transfer (5-7). These examples highlight two major in immunoblots which was previously not detectable with any of the commonly used blotting procedures. factors influencing the efficiency of protein blotting: the Since the improved conditions offer increased sensi-elution efficiency of a protein out of a given gel matrix tivity across a spectrum of protein sizes, they should and the efficiency of binding by the membrane. Imbe widely applicable. ᭧ 1997 Academic Press
provements in one area often appear to be gained at some cost in the other; methanol has been reported to increase the binding capacity of nitrocellulose, perhaps through hydrophobic effects (8), but it may decrease the The electrophoretic transfer of proteins from polyelution efficiency of large proteins by partially ''fixing'' acrylamide gels to nitrocellulose sheets, initially dethem in the gel (for review, see 9). scribed by Towbin (1), has enjoyed widespread use as Recently, our efforts to study a large (ca. 500 kDa) a valuable tool in the field of protein research. One membrane protein were stymied by our inability to deapplication of particular import has been the subsetect the protein on immunoblots. Suspecting that this quent employment of antibody probes directed toward reflected a poor elution efficiency from the gel, we emthe nitrocellulose-bound proteins. Variously termed barked on a systematic study of some of the parameters immunoblotting, protein blotting, or ''Western'' blotting