In mammals, circadian clocks are present in a variety of tissues and cells, and these clocks are classified as the central clock and peripheral clocks. Peripheral clocks are present in various tissues, such as heart, liver and kidney, and are mainly reset by feeding timing and various hormones. However, the synchronization mechanisms of individual peripheral clocks via hormones and their receptors remain unclear. In our previous studies, Multi-PK antibodies (M1C, M8C and YK34) which directed to a highly conserved region of protein kinases were developed. M1C and M8C antibodies recognize a wide variety of serine/threonine protein kinases and YK34 antibody detects tyrosine protein kinases. In this study, we analyzed diurnal variation of tyrosine kinases in cell membrane fractions from mouse peripheral tissues by Western blotting using YK34 antibody. Diurnal variation of tyrosine kinase expression was detected in heart, liver, and kidney. In the liver, the 180-kDa and 130-kDa bands that changed diurnally were identified as epidermal growth factor receptor (EGFR). EGFR was not expressed in other peripheral tissues. These results suggest that EGFR may contribute to the synchronization of the circadian clock in mouse liver.

Enzymes retain their activity even after separation by nondenaturing two-dimensional electrophoresis (2DE), along with fluorescent substances that can associate and dissociate from the enzymes. Esterase and sorbitol dehydrogenase, which retain their activity, can be reseparated by nondenaturing electrophoresis following the separation of mouse liver cytosolic proteins by nondenaturing 2DE. Fluorescent detection was achieved using enzymatic products such as 4-methylumbelliferone and the reduced form of nicotinamide adenine dinucleotide (NADH). Moreover, the activities of these two enzymes could be quantitatively reanalyzed even after separation via nondenaturing 2DE and fluorescent detection. The activities of esterase and sorbitol dehydrogenase were considerably suppressed by 1 mM Fe²⁺, reducing to 0.24-fold and 0.28-fold of their original levels, respectively. After washing out of Fe²⁺, esterase activity remained suppressed, further decreasing to 0.14-fold, whereas sorbitol dehydrogenase activity increased to 1.31-fold of its original suppressed level. These findings demonstrate that the current method of reseparating and reanalyzing enzymes is suitable for investigating the reversibility or irreversibility of enzyme activity in response to factors such as metal ions.

We previously demonstrated that agarose native gel electrophoresis using a 0.1 M histidine (His)/0.1 M MES buffer (pH 6.1) is effective for separating various proteins regardless of their charged states. In this study, we compared the performance of this buffer with different buffer systems: 0.1 M His/0.1 M MOPS (pH 6.6), 0.1 M imidazole/0.1 M MOPS (pH 7.2), 0.1 M imidazole/0.1 M HEPES (pH 7.4), 25 mM Tris/192 mM Glycine (pH 8.5) and 1×TAE (40 mM Tris-acetate, 1 mM EDTA) (pH 8.3). Using acidic BSA, neutral IgG, and basic lysozyme as model proteins, electrophoresis was conducted under identical conditions: 1% (w/v) agarose, horizontal electrophoresis, a constant voltage of 100 V, and the same run time. Among the tested systems, the 0.1 M His/0.1 M MES buffer (pH 6.1) demonstrated superior performance, providing balanced separation and clear detection of all three proteins. In contrast, while acidic BSA migrated successfully in all cases, IgG showed little migration and lysozyme displayed band tailing, smearing, or unexpectedly fast migration in the other buffer systems. Additionally, three IgG proteins with varying isoelectric points achieved optimal separation with the 0.1 M His/0.1 M MES buffer, further highlighting its effectiveness for diverse protein analyses.

Common isoforms of apolipoprotein E (apoE) can be distinguished into apoE2, E3, and E4. Isoforms apoE2 and apoE4 are associated with dyslipidemia, while apoE4 is associated with the development of Alzheimer’s disease. ApoE mutation analysis is often performed solely through genetic testing. However, because charge-differentiated mutations frequently impact the biological activity of apoE, isoelectric focusing (IEF) serves as a critical method for protein charge analysis. The apoE phenotype determined by IEF immunoblotting, which allows simultaneous electrophoresis of 48 samples, was compared with apoE genotypes in 815 Japanese individuals. The results demonstrated a 97.6% match rate between apoE phenotype and genotype, with 795 of 815 cases matching. IEF immunoblotting revealed 3 apoE2/2, 577 E3/3, 11 E4/4, 61 E2/3, 14 E2/4, 130 E3/4, 3 E3/5, 1 E4/5, 1 E2/7, 12 E3/7, and 2 E4/7. Comparison with the restriction enzyme polymorphism assay using HhaI, which detects only genotypes ε2, ε3, and ε4, indicated that deviations were primarily due to apoE5 and E7, with one exception. One discordant case has phenotype E3/4 and genotype ε3/3. Sanger sequencing of the APOE gene identified a novel heterozygous APOE4 variant (Pro295Arg, p.Pro313Arg). This individual exhibited normolipidemia, with a serum total cholesterol level of 153 mg/dL, a high-density lipoprotein cholesterol level of 85 mg/dL, and a triglyceride level of 47 mg/dL. The IEF immunoblotting method, which permits simultaneous comparison of multiple samples, is a valuable technique for determining apoE phenotypes.

A rapid detection and identification method for viruses based on variations in their structural proteins (SP) was developed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Viral SPs purified by ultracentrifugation using sucrose were cleaved by formic acid (FA) with heating (30 min) or microwaving (5 min). The resulting peptides were analysed using MALDI-TOF MS in a linear mode with a α-cyano-4-hydroxy cinnamic acid matrix. Peptide mass fingerprints (PMF) from SPs were compared, and viruses were identified using data matching analysis. All analysed viruses, insect and animal viruses, showed unique PMFs. Viruses could be detected and identified based on differences in PMFs obtained from SPs after quick FA treatment with heating or microwaving. Short FA cleavage of SPs by heating or microwaving against purified virus SPs using ultracentrifugation enabled rapid and cost-effective detection and identification of viruses.