ANALYTICAL BIOCHEMISTRY 250, 61–65 (1997) ARTICLE NO. AB972196 Micropreparative High Resolution Purification of Proteins by a Combination of Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis, Isoelectric Focusing, and Membrane Blotting Fang Ting Liang,* David E. Granstrom,* John F. Timoney,* ,1 and Yu Fang Shi† *Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, Kentucky 40546; and †Department of Immunology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855 Received November 4, 1996 8) chromatography, are used when a large amount of We report a simple, economical, and efficient proto- sample is available. However, in many situations the col for protein purification from cells. First, proteins quantity of samples is limited and recovery during of cell lysates were separated by standard sodium do- chromatographic processes is often inadequate. In ad- decyl sulfate – polyacrylamide gel electrophoresis dition, chromatographic methods result in a large (SDS – PAGE) and electroblotted to protein-blotting array of fractions, which need to be screened for target membrane. The blots were stained with Coomassie proteins. blue or developed by immunoblotting to visualize spe- In contrast to chromatographic methods, electropho- cific proteins. The bands corresponding to those visi- retic techniques, such as isoelectric focusing (IEF) 2 and ble by immunoblotting were excised from the dye- sodium dodecyl sulfate – polyacrylamide gel electropho- stained blots and subjected to isoelectric focusing. The resis (SDS – PAGE) provide high resolution and require focused gel was stained with Coomassie blue. Finally, a smaller amount of sample (9, 10). For example, IEF the stained bands were excised and subjected to an- can separate proteins differing in their isoelectric point other SDS – PAGE separation and electrotransferred (pI ) values by as little as 0.02 pH units, and SDS– back to protein-blotting membrane. At this stage, the PAGE can resolve proteins differing by 0.5 kDa. Com- purified proteins were suitable for microsequencing. bination of these two techniques results in a high-reso- We have tested the feasibility of this novel technique lution two-dimensional polyacrylamide gel electropho- by purifying proteins with molecular weights ranging retic technique (2D-PAGE), which can separate a from 19 to 100 kDa from a lysate of Sarcocystis neu- complex protein mixture based on both pI and molecu- rona, the etiologic agent of equine protozoal mye- lar weight (11). However, it is not a good preparative loencephalitis. The purity of proteins was demon- protocol because of the low loading capacity both in strated by reverse-phase high-performance liquid chromatography. Partial sequences of these purified terms of sample volume and protein amount. Moreover, proteins were obtained by N-terminal or digestive se- as an analytical method, protein bands resolved by the quencing.  1997 Academic Press second-dimensional gel are not sharp but diffuse, ne- gating some separation already obtained by the first- dimensional gel electrophoresis. The procedure of 2D- PAGE is also weakened by masking of trace proteins Although many techniques have been developed for by more abundant proteins, especially when a crude protein purification, most of the intensive procedures protein extract is analyzed. do not meet the practical requirements of most biomed- ical research laboratories. Chromatographic tech- 2 Abbreviations used: IEF, isoelectric focusing; SDS – PAGE, so- niques, such as ion-exchange (1, 2), gel filtration (3), dium dodecyl sulfate – polyacrylamide gel electrophoresis; pI, isoelec- tric point; 2D-PAGE, high-resolution two-dimensional polyacryl- hydroxyapatite (4), hydrophobic (5, 6), or affinity (7, amide gel electrophoretic technique; NC, nitrocellulose; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; RP – HPLC, reverse-phase high-performance liquid chromatography; 1 To whom correspondence should be addressed. Fax: (606) 257- 4172. TFA, trifluoroacetic acid. 61 0003-2697/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.