Abstract
A method of direct tissue isoelectric focusing (DTIF) in agarose of human skeletal muscle is described. This particular method was developed to utilize the small amounts of tissue obtained by needle muscle biopsies performed for diagnostic purposes. 20 ΞΌm thick cryostat sections were adhered to the hydrophilic surfaces of small GelBond rectangles. These were then applied directly to the surface of the gels. A chargeβbalanced purified agarose was used to make the gels, which contained Triton Xβ100 to enhance protein solubilization. Stabilization of the pH gradient was attempted by employing a 3% w/v ampholyte which was a blend of 0.85% w/v pH 8β10.4 and 2.15 % w/v pH 3β10 Pharmalyte, by using anolyte regulation with 0.1 M aspartic acid and by focusing the gels under a CO~2~βextracted nitrogen atmosphere. Equilibrium of proteins stained by Coomassie Brilliant Blue Rβ250 was apparent as monitored by comigration of sections from both anode and cathode. With the exception of the cathode end, excellent resolution and reproducibility was achieved. Better cathode resolution was noted with nonβequilibirium conditions. Preliminary zymograms of lactic dehydrogenase (LDH), using a tetrazolium technique, have shown consistent patterns of multiple isoenzymes, the basic components of which were best seen using nonβequlibirium conditions.