ANALYTICALBIOCHEMISTRY 179,19&197 (1989) Visualization of Acid Phosphatase Activity on Nitrocellulose Filters following Electroblotting of Polyacrylamide Gels’ Angelos K. Kanellis, **2Theophanes Solomos,* and Autar K. Mattoot *Department of Horticulture, University of Maryland, College Park, Maryland 20742, and tPlant Hormone Laboratory, USDAIARS, Beltsville Agricultural Research Center (W), Beltsville, Maryland 20705 Received August 26,19&3 A method for visualizing acid phosphatase isoen- zymes by activity staining on nitrocellulose filters after electroblotting of proteins fractionated on nondenatur- ing polyacrylamide gels is described. Reproducible re- sults were obtained when 25 mM Tris-192 mM glycine was used as the transfer buffer instead of 0.7% acetic acid, 60 mM sodium acetate, pH 4, or 0.14 M acetic acid- 0.35 M &alanine, pH 4.3. Dot-blot analysis of banana fruit extracts on nitrocellulose filters revealed that a minimum of 6 X lo-’ units (nmol p-nitrophenyl phos- phate hydrolyzed. g-’ .h-‘) of acid phosphatase activity can be detected. This method can be suitable for screen- ing a large number of biological samples for monitoring acid phosphatase activity. o 1989 Academic PW. hc. Isoenzymes as biochemical markers are useful in stud- ies encompassing breeding, evolution, and development of plants. A number of isoenzymes have been shown to vary in amount and activity during ripening of fruits (1,15). In particular, the activity of acid phosphatase as- sociated with either the soluble or the particulate cell fractions markedly increases during ripening (2,3). In this report, we describe a method for the direct visualiza- tion of acid phosphatase activity on nitrocellulose filters following fractionation of banana proteins in nondena- turing polyacrylamide gels. MATERIALS AND METHODS Extraction and polyacrylamide gel electrophoresis (PAGE)3 of acid phosphatase (AP) isoenzymes. One 1 Scientific Article No A-4919, Contribution No 7962 of the Mary- land Agricultural Experiment Station. ’ Present address: Institute of Molecular Biology and Biotechnol- ogy, Foundation of Research and Technology-Hellas, P.O. Box 1570, 714 09 Iraklion, Crete, Greece. 3 Abbreviations used: PAGE, polyacrylamide gel electrophoresis; AP, acid phosphatase; ABS, acetate-buffered saline; PBS, phosphate- 194 gram of frozen pulverized banana pulp tissue was mixed with 1.5 ml of 50 mM Tris-HCl, pH 7.5, containing 5 mM B-mercaptoethanol, 0.5 mM phenylmethanesulfonyl fluoride (PMSF), 10 ~.LM leupeptin, 1 mM diethyldithio- carbamic acid, 5% (w/w fresh wt) polyvinylpolypyrroli- done, 1 mM EDTA, and 10% glycerol. After thawing on ice, total proteins were solubilized by vortexing the mix- ture three times for 5 s at 5-min intervals during which time the samples were left on ice. Following centrifuga- tion at 21,000g for 60 min, the supernatant was saved and served as the soluble fraction. The pellet was resus- pended in 1 ml of 50 mM Tris-HCl, pH 7.5, containing 5 mM @mercaptoethanol, 0.5 mM PMSF, 10 PM leupeptin, 1 mM diethyldithiocarbamic acid, 1 mM EDTA, 10% (v/ v) glycerol, 0.5 M NaCl, and 0.5% (v/v) Triton X-100. After vortexing as above, the mixture was centrifuged at 21,000g for 60 min. The supernatant served as the solu- bilized particulate fraction. Both soluble and particulate fractions were recentrifuged at 45,000 rpm for 60 min in a Beckman4 50 Ti rotor and the clear supernatants obtained were divided into 0.5-ml aliquots and stored at -70°C until used. In preliminary experiments, banana acid phosphatase isoenzymes were found to move toward the anode in a 1% agarose gel in a 0.155 M Tris-0.043 M citric acid, pH 7, continuous buffer system (4), indicative of proteins with ~1’s lower than 7. However, in this buffer system or the high pH system (5), acid phosphatase isoenzymes were poorly resolved. The method of choice was found to be that of Reisfeld et al. (6). The pH of the stacking gel buffer was adjusted to 6.8 and that of the resolving gel to 4.3. The polarity of leads in the electrophoretic appara- tus was reversed, and 1% methylene green was used as buffered saline; PMSF, phenylmethanesulfonyl fluoride; BSA, bovine serum albumin. 4 Mention of a company name or trademark does not constitute en- dorsement by the U.S. Department of Agriculture over others of a sim- ilar nature not mentioned. 0003-2697/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.