Detection and characterization of epidermal proteinases by polyacrylamide gel electrophoresis

Electrophoresis of cornified cell extracts of 2-day-old rats, using SDS polyacrylamide gels copolymerized with alpha-casein or gelatin with or without plasminogen, was performed. Both Tris-HCl buffer soluble protein and KSCN solubilized protein contained a number of hydrolases for alpha-casein and/or gelatin, but PA (mol. wts 57 and 50K) was found only in the KSCN extract. The pH dependency, substrate specificity and mol. wt of plasminogen-independent proteinases were comparatively determined and DFP inhibition tested. This simple technique helped to identify the presence of several proteinases and to characterize them in partially purified epidermal extracts.     

Rapid microsatellite analysis using discontinuous polyacrylamide gel electrophoresis.

A method for screening large numbers of samples for microsatellites using discontinuous, non-denaturing polyacrylamide gels and rapid fluorescent gel staining is described. Disc electrophoresis on slab gels provides high-resolution of PCR products. It is useful for collecting population data once microsatellite loci have been characterized.     

Apolipoprotein distribution in human lipoproteins separated by polyacrylamide gradient gel electrophoresis

The heterogeneity of serum lipoproteins (excluding very low density (VLDL) and intermediate density (IDL) lipoproteins) and that of lipoproteins secreted by HepG2 cells has been studied by immunoblot analysis of the apolipoprotein composition of the particles separated by polyacrylamide gradient gel electrophoresis (GGE) under nondenaturing conditions. The reactions of antibodies to apoA-I, apoA-II, apoE, apoB, apoD, and apoA-IV have revealed discrete bands of particles which differ widely in size and apolipoprotein composition. GGE of native serum lipoproteins demonstrated that apoA-II is present in lipoproteins of limited size heterogeneity (apparent molecular mass 345,000 to 305,000) and that apoB is present in low density lipoproteins (LDL) and absent from all smaller or denser lipoproteins. In contrast, serum apoA-I, E, D, and A-IV are present in very heterogeneous particles. Serum apoA-I is present mainly in particles of 305 to 130 kDa where it is associated with apoA-II, and in decreasing order of immunoreactivity in particles of 130-90 kDa, 56 kDa, 815-345 kDa, and finally within the size range of LDL, all regions where there is little detectable apoA-II. Serum apoE is present in three defined fractions, one within the size range of LDL, one containing heterogeneous particles between 640 and 345 kDa, and one defined fraction at 96 kDa. Serum apoD is also present in three defined fractions, one comigrating with LDL, one containing heterogeneous particles between 390 and 150 kDa, and one band on the migration front. Most of serum apoA-IV is contained in a band comigrating with albumin. GGE of centrifugally prepared LDL shows the presence of apoB, apoE, and apoD, but not that of apoA-I. However, the particles containing apoA-I, which, in serum, migrated within the LDL size range and as bands of 815 to 345 kDa, were recovered upon centrifugation in the d greater than 1.21 g/ml fraction. GGE of high density lipoproteins (HDL) indicated that most of apoA-I, A-II, and A-IV were present in lipoproteins of the same apparent molecular mass (390-152 kDa). ApoD tended to be associated with large HDL, and this was also significant for HDL apoE, which is present in lipoproteins ranging from 640 to 275 kDa. GGE of very high density lipoproteins (VHDL) presented some striking features, one of which was the occurrence of apolipoproteins in very discrete bands of different molecular mass. ApoA-II was bimodally distributed at 250-175 kDa and 175-136 kDa, the latter fraction also containing apoA-I.(ABSTRACT TRUNCATED AT 400 WORDS)     

Separation of aminoacyl-transfer RNAs by polyacrylamide gel electrophoresis

A polyacrylamide gel electrophoresis system for separating $\text{E.}$$\text{coli}$ tRNAs and aminoacyl-tRNAs is described. The tRNA was separated into 6 discrete bands which contained varyin aamounts of tRNA and therefore varying numbers of tRNA species. In order to locate specific tRNAs, tRNA was charged with a ¹⁴C amino acid and the aminoacyl-tRNA was located by autoradiography. With several amino acids, 2 isoaccepting species were found. In total, 30 aminoacyl-tRNAs were located.     

Structural analysis of glycopeptides by polyacrylamide gel electrophoresis

Three different well-characterized preparations of proteoglycan subunits were analyzed by high-performance liquid chromatography on a silica-based material bonded with an amide phase. The biochemical integrity of the proteoglycan subunits was retained during this procedure. The high sensitivity coupled with the increased speed of high-performance liquid chromatography will permit rapid analysis and comparisons of very small specimens.     

Separation of t-RNAs by two-dimensional polyacrylamide gel electrophoresis

A pH 5.8 polyacrylamide gel electrophoresis buffer is described. Electrophoresis in this MES-citrate system at pH 5.8 separates E. coli transfer RNAs into 15 bands using 15% acrylamide gels. Polyacrylamide gel electrophoresis in a second dimension at pH 8.3 further resolves E. coli t-RNAs into 20 spots.     

Nucleotide composition of RNA by polyacrylamide gel electrophoresis

A procedure using polyacrylamide gel electrophoresis has been devised for determining the nucleotide composititon of small amounts (50 μg) of RNA. The procedure permits analysis of the separated nucleotides in the gel matrix, thereby avoiding sample manipulation and allowing for greater reproducibility.     

Studies on mitochondrial proteins I. Separation and characterization by polyacrylamide gel electrophoresis

Rat liver mitochondria were fractionated into inner and outer membranes and soluble intermembrane space and matrix. The protein components of these fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mitochondria contained at least 20 components ranging in molecular weights from 10 000 to 140 000. Inner membranes differed markedly from outer membranes both in number of components and size distribution. The intermembrane space contained a few polypeptide species. These were of low molecular weight. The matrix was characterized by a high molecular weight component (130 000) which comprised 30% of this fraction. A major carbohydrate-containing polypeptide with an approximate molecular weight of 93 000 was detected in outer membrane preparations.     

High concentration polyacrylamide gel electrophoresis

A simple technique is described for easy removal of high concentration polyacrylamide gels after electrophoresis from glass tubes without breaking them.