Electrophoresis zyxwvutsrqpo 1981,2, 25 1-258 Direct tissue isoelectric zyxw focusing of muscle 25 1 Basil J. Thompson, Arthur H. M. Burghes, Michael J. Dunn and Victor Dubowitz Jerry Lewis Muscle Research Centre, Department of Paediatrics and Neonatal Medicine, Hammersmith Hospital, London The application of direct tissue isoelectric focusing to the study of human skeletal muscle 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 pur- poses. 20 pm 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 charge-balanced purified agarose was used to make the gels, which contain- ed Triton X-100 to enhance protein solubilization. Stabilization of the pH gradient was attempted by employing a 3 zyxwv % w/v ampholyte which was a blend of 0.85 % wlv pH 8-10.4 and 2.15 % w/v pH 3-10 Pharmalyte, by using anolyte regulation with 0.1~ aspartic acid and by focusing the gels under a COz-extracted nitrogen at- mosphere. Equilibrium of proteins stained by Coomassie Brilliant Blue R-250 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 non-equlibrium conditions. Preliminary zymograms of lactic dehydrogenase (LDH), using a tetrazolium technique, have shown consistent patterns of multiple isoenzymes, the basic com- ponents of which were best seen using non-equlibrium conditions. 1 Introduction The small amount of tissue available from clinical biopsy material can place considerable constraints on the scope of the biochemical analyses that can be performed. Such is the case with the amount of muscle tissue available from biopsies of patients undergoing investigation for possible muscle dis- orders. A method which is suitable for small amounts of tissue has been described by Saravis zyxwvuts et zyxwvutsrq al. [ll. This involves DTIF in agarose of 4-8 pm thick tissue sections of fresh frozen samples of colonic adenocarcinoma. Furthermore, Saravis 111 has shown that extraction by DTIF does not result in the loss or reduction of bands that occurs when saline extracts of homogenized tissue sections are focused. Whether this effect is due to denaturation, proteolysis or other factors, direct extraction would appear to be preferable to tissue homogenization. This might be particularly relevant to muscle tissue given the presence of endogenous proteases and acid hydrolases [2,31 and the higher concentrations of these that have been detected in diseased muscle [41. When electrophoretic techniques are applied to muscle, the abun- dant structural proteins often obscure the quantitatively minor soluble proteins. Giometti et al. [51 have approched this problem by employing high-resolution two-dimensional gel electrophoresis. However, this method uses more tissue (100-200 mg) than is usually available from needle muscle biopsies [61, involves tissue homogenization and precludes zymogram analysis due to protein denaturation. Therefore, as DTIF seems well-suited to the study of soluble muscle proteins, we have developed a method of DTIF in Correspondence: Basil J. Thompson, Jerry Lewis Muscle Research Centre, Hammersmith Hospital, Du Cane Road, London W12 zyxwvut OHS, England Abbreviations: BHT: Butylated hydroxy-toluene; DMD: Duchenne muscular dystrophy; DTIF: Direct tissue isoelectric focusing; IEF: Isoelectric focusing; LDH: Lactic dehydrogenase; P-NAD: p- Nicotinamide adenine dinucleotide; NBT: Nitro blue tetrazolium; PI: Isoelectric point; PMS:Phenazine methosulphate; SDS: Sodium dodecyl sulphate; TCA: Trichloroacetic acid 0 Verlag Chemie GmbH, D-6940 Weinheim, 1981 agarose which utilizes cryostat sections from diagnostic needle muscle biopsies. The method employs a broad pH gradient with modified conditions to enhance anodic and cathodic resolution in order to optimize protein maps for a proposed comparative analysis of samples from normal and diseased muscle. Equilibrium conditions have been defined. Moreover, the method can be coupled with an analysis of non-soluble muscle proteins and lends itself readily to zymogram analysis of muscle enzymes. 2 Materials and methods 2.1 Chemicals Except where specifically indicated, chemicals and reagents used were obtained from either BDH Chemicals Limited (Poole, England) or Sigma Chemical Corporation (St. Louis, USA). Deionized distilled water was used to prepare the gels and electrolyte solutions. 2.2 Sample preparation Needle muscle biopsies were obtained from the quadriceps of patients undergoing investigation for possible muscle dis- orders by the Bergstrom needle technique [6-81. Biopsies were kept sterile on normal saline (0.9 % w/v NaC1) soaked gauze for up to 1 h before processing. The pieces were then orientated on cork 181, a small amount of OCT Compound (Lab Tek Products, Miles Laboratories Incorporated, 11- linois, USA) was placed around the tissue and the entire assembly immersed for 20-30 s in liquid CClzF2 (Arcton 12, Imperial Chemical Industries Limited, Cheshire, England) cooled in liquid nitrogen. Tissue sections were obtained using a Slee Standard Duty Model Cryostat type HR (Slee Medical Equipment Limited, London) operated at -20 "C. First, 10 ym sections were obtained for routine histopathology, and then 20 pm sections were cut for isoelectric focusing (IEF). GelBond (0.1 mm thickness, Marine Colloids Divi- sion, Rockland, Maine, USA) strips were prepared measur- 0173-0835/81/0410-025 1 S2.50/0