Electrophoresis, a transport technology that transitioned from moving boundary method to zone method.

Gel electrophoresis, a transport technology, is one of the most widely used experimental methods in biochemical and pharmaceutical research and development. Transport technologies are used to determine hydrodynamic or electrophoretic properties of macromolecules. Gel electrophoresis is a zone technology, where a small volume of sample is applied to a large separation gel matrix. In contrast, a seldom-used electrophoresis technology is moving boundary electrophoresis, where the sample is present throughout the separation phase or gel matrix. While the zone method gives peaks of separating macromolecular solutes, the moving boundary method gives a boundary between solute-free and solute-containing phases. We will review electrophoresis as a transport technology of zone and moving boundary methods and describe its principles and applications.

Development of a novel two-dimensional gel electrophoresis protocol with agarose native gel electrophoresis.

A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native-PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS-PAGE gels or the edge of the flat SDS-MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen-antibody complexes, as well as complex proteins such as IgM pentamer and ß-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5-6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.

Detection of concentration-dependent conformational changes in SARS-CoV-2 nucleoprotein by agarose native gel electrophoresis.

The nucleoprotein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is abundantly expressed during infection, making it a diagnostic target protein. We analyzed the structure of the NP in solution using a recombinant protein produced in E. coli. A codon-optimized Profinity eXact™-tagged NP cDNA was cloned into pET-3d vector and transformed into E. coli T7 Express. The recombinant protein was first purified via chromatographic step using an affinity tag-based system that was followed by tag cleavage with sodium fluoride, resulting in proteolytic removal of the N-terminal tag sequence. The digested sample was then loaded directly onto a size exclusion chromatography run in the presence of L-Arg-HCl, resulting in removal of host nucleic acids and endotoxin. The molecular mass of the main NP fraction was determined by mass photometry as a dimeric form of NP, consistent with the blue native PAGE results. Interestingly, analysis of the purified NP by our newly developed agarose native gel electrophoresis revealed that it behaved like an acidic protein at low concentration despite its alkaline isoelectric point (theoretical pI = 10) and displayed a unique character of concentration-dependent charge and shape changes. This study should shed light into the behavior of NP in the viral life cycle.

Analysis of bovine serum albumin unfolding in the absence and presence of ATP by SYPRO Orange staining of agarose native gel electrophoresis.

An attempt was made to specifically stain unfolded proteins on agarose native gels. SYPRO Orange is routinely used to detect unfolded protein in differential scanning fluorimetry, which is based on the enhanced fluorescence intensity upon binding to the unfolded protein. We demonstrated that this dye barely bound to the native proteins, resulting in no or faint staining of the native bands, but bound to and stained the unfolded proteins, on agarose native gels. Using bovine serum albumin (BSA), it was shown that staining did not depend on whether BSA was thermally unfolded in the presence of SYPRO Orange or stained after electrophoresis. On the contrary, SYPRO Orange dye stained protein bands in the presence of sodium dodecylsulfate (SDS) due to incorporation of the dye into SDS micelles that bound to the unfolded proteins. This staining resulted in detection of new, intermediately unfolded structure of BSA during thermal unfolding. Such intermediate structure occurred at higher temperature in the presence of ATP.

[A Case of Extranodal NK/T-Cell Lymphoma with Perforation of the Small Intestine].

A rare case of extranodal NK/T-cell lymphoma(ENKL)with small intestinal perforation is reported. A 92-year-old man was admitted for a loss of consciousness. Computed tomography(CT)scan revealed the presence of an intraperitoneal abscess that was drained. Two days later, the drained fluid changed to intestinal juice, and intestinal perforation was suspected. The patient underwent surgery which revealed a 1 cm perforation site in the ileum. A high fever continued after surgery, and malignant lymphoma was diagnosed from pathological findings; however, further treatment could not be performed. He died 24 days after the operation. Pathological dissection revealed metastasis of ENKL at the systemic lymph nodes.

Machine Learning to Predict the Rapid Growth of Small Abdominal Aortic Aneurysm.

OBJECTIVE: The purpose of this study was to determine whether computed tomography (CT) angiography with machine learning (ML) can be used to predict the rapid growth of abdominal aortic aneurysm (AAA). MATERIALS AND METHODS: This retrospective study was approved by our institutional review board. Fifty consecutive patients (45 men, 5 women, 73.5 years) with small AAA (38.5 ± 6.2 mm) had undergone CT angiography. To be included, patients required at least 2 CT scans a minimum of 6 months apart. Abdominal aortic aneurysm growth, estimated by change per year, was compared between patients with baseline infrarenal aortic minor axis. For each axial image, major axis of AAA, minor axis of AAA, major axis of lumen without intraluminal thrombi (ILT), minor axis of lumen without ILT, AAA area, lumen area without ILT, ILT area, maximum ILT area, and maximum ILT thickness were measured. We developed a prediction model using an ML method (to predict expansion >4 mm/y) and calculated the area under the receiver operating characteristic curve of this model via 10-fold cross-validation. RESULTS: The median aneurysm expansion was 3.0 mm/y. Major axis of AAA and AAA area correlated significantly with future AAA expansion (r = 0.472, 0.416 all P < 0.01). Machine learning and major axis of AAA were a strong predictor of significant AAA expansion (>4 mm/y) (area under the receiver operating characteristic curve were 0.86 and 0.78). CONCLUSIONS: Machine learning is an effective method for the prediction of expansion risk of AAA. Abdominal aortic aneurysm area and major axis of AAA are the important factors to reflect AAA expansion.

The complete mitochondrial genome of Sarcoptes scabiei var. nyctereutis from the Japanese raccoon dog: Prediction and detection of two transfer RNAs (tRNA-A and tRNA-Y).

Sarcoptes scabiei (Acari: Sarcoptidae) causes a common contagious skin disease that affects many mammals. Here, the complete mitochondrial genome of a mite, S. scabiei var. nyctereutis, from Japanese wild raccoon dogs was analyzed. The 13,837bp circular genome contained 13 protein-coding genes, two rRNA genes, and 22 tRNA genes. For the first time, two tRNAs (alanine and tyrosine), that were thought to be absent in scabies mites from other animals, were predicted to have short, non-cloverleaf structures by in silico annotation and detected by RT-PCR, sequencing, and northern analysis. The mitochondrial genome structure of S. scabiei is similar to that of Psoroptes cuniculi and Dermatophagoides farinae. While small and unusual tRNA genes seem to be common among acariform mites, further experimental evidence for their presence is needed. Furthermore, through an analysis of the cox1 gene, we have provided new evidence to confirm the transmission of this mite between different animal hosts.

Simultaneous acquisition of MR angiography and diagnostic images of abdomen at view-sharing multiarterial phases and comparing the effect of two different contrast agents.

BACKGROUND: Simultaneous acquisition of magnetic resonance angiography (MRA) and diagnostic images is challenging in contrast-enhanced upper abdominal MRI. PURPOSE: To evaluate the image quality of MRA of the abdomen acquired simultaneously with diagnostic MR images, and to compare the contrast effect, conspicuity of aortic branches, and pancreatic lesions in MRA between gadobutrol and gadoterate meglumine. STUDY TYPE: Prospective. POPULATION: Eighty-eight patients with known and suspected upper abdominal disease. FIELD STRENGTH/SEQUENCES: 3T/4D-eTHRIVE (T1 -weighted fat-suppressed 3D fast gradient echo) for multiarterial phase imaging. ASSESSMENT: The artery-to-muscle signal intensity ratio (SIR), conspicuity of aortic branches on the axial, maximum intensity projection (MIP), and volume-rendered (VR) images, and conspicuity of focal pancreatic lesions were compared between gadobutrol and gadoterate meglumine. The diameters of aortic branches were measured on axial MRA and computed tomography angiography (CTA) images and then compared. STATISTICAL TESTS: Quantitative and qualitative data were assessed with the Mann-Whitney U-test. The diameters of aortic branches between MRA and CTA were compared with a Spearman rank correlation test. RESULTS: View-sharing multiarterial phase imaging was successfully performed in all patients. The SIRs of common hepatic artery (P = 0.0051) and left renal artery (RA) (P = 0.045), vascular conspicuities of right and left hepatic arteries (P = 0.010 and 0.030) and right and left RAs on axial (P = 0.0065 and 0.036), and that of gastroduodenal artery on MIP (P = 0.039) with gadobutrol were significantly higher than those with gadoterate meglumine. The conspicuity of focal pancreatic lesions were comparable between the gadobutrol and gadoterate meglumine (P = 0.73). The vascular diameters on MRA and CTA were strongly correlated in all aortic branches (r = 0.842-0.942, P < 0.0001). DATA CONCLUSION: High-quality MRA of the abdomen was obtained simultaneously with the diagnostic MR images using view-sharing multiarterial phase imaging that also demonstrated comparable image quality between gadobutrol and gadoterate meglumine. LEVEL OF EVIDENCE: 2 Technical Efficacy Stage 1 J. Magn. Reson. Imaging 2017.

Improving the quality of a recombinant rabbit monoclonal antibody against PLXDC2 by optimizing transient expression conditions and purification method.

Rabbit monoclonal antibodies (mAbs) have many advantages over mouse antibodies in biological research and diagnostics applications because they exhibit high affinity and specificity. However, the methods of recombinant rabbit mAb production have not been optimized to the same extent as techniques used to produce mouse and human mAbs. In this study, we sought to optimize the production of a recombinant rabbit mAb against human plexin domain containing protein 2 (PLXDC2), a known cell surface antigen, by culturing HEK293-6E cells transfected with antibody-encoding genes at two different temperatures and by purifying the end-product by three different chromatography methods. The quality and function of purified antibody preparations were checked by electrophoresis and western blot analysis. The secreted rabbit mAb produced by a combination of culturing at 32 °C, purification by ammonium sulfate fractionation, and diethylaminoethyl resin (DEAE) ion exchange chromatography was of high quality. In contrast, the antibody produced by the cells grown at 37 °C for 6 days after transfection and purified by Protein A/G affinity method was low quality. Hypothermic conditions during production reduced protein heterogeneity probably by favorably affecting the levels of glycosylation and aggregation. In particular, according to western blotting data, CIMmultus DEAE chromatography that utilizes monolithic columns not only excluded inferior charge variants resulting from nonspecific reactions but also yielded rabbit mAb that was of better quality than commercially available rabbit polyclonal antibodies. The combination of techniques suggested by us may be a general approach to enhance product quality of rabbit mAbs produced by transient expression systems.

Dual-layer DECT for multiphasic hepatic CT with 50 percent iodine load: a matched-pair comparison with a 120 kVp protocol.

OBJECTIVES: To evaluate the image quality and lesion conspicuity of virtual-monochromatic-imaging (VMI) with dual-layer DECT (DL-DECT) for reduced-iodine-load multiphasic-hepatic CT. METHODS: Forty-five adults with renal dysfunction who had undergone hepatic DL-DECT with 300-mgI/kg were included. VMI (40-70-keV, DL-DECT-VMI) was generated at each enhancement phase. As controls, 45 matched patients undergoing standard 120-kVp protocol (120-kVp, 600-mgI/kg, and iterative reconstruction) were included. We compared the size-specific dose estimate (SSDE), image noise, CT attenuation, and contrast-to-noise ratio (CNR) between protocols. Two radiologists scored the image quality and lesion conspicuity. RESULTS: SSDE was significantly lower in DL-DECT group (p < 0.01). Image noise of DL-DECT-VMI was almost constant at each keV (differences of ≤15%) and equivalent to or lower than of 120-kVp. As the energy decreased, CT attenuation and CNR gradually increased; the values of 55-60 keV images were almost equivalent to those of standard 120-kVp. The highest scores for overall quality and lesion conspicuity were assigned at 40-keV followed by 45 to 55-keV, all of which were similar to or better than of 120-kVp. CONCLUSIONS: For multiphasic-hepatic CT with 50% iodine-load, DL-DECT-VMI at 40- to 55-keV provides equivalent or better image quality and lesion conspicuity without increasing radiation dose compared with standard 120-kVp protocol. KEY POINTS: • 40-55-keV yields optimal image quality for half-iodine-load multiphasic-hepatic CT with DL-DECT. • DL-DECT protocol decreases radiation exposure compared with 120-kVp scans with iterative reconstruction. • 40-keV images maximise conspicuity of hepatocellular carcinoma especially at hepatic-arterial phase.

Characterization of Liver Tumors by Diffusion-Weighted Imaging: Comparison of Diagnostic Performance Using the Mean and Minimum Apparent Diffusion Coefficient.

PURPOSE: To determine the minimum apparent diffusion coefficient (ADC(min)) values of benign and malignant hepatic lesions based on diffusion-weighted imaging and to compare the diagnostic performance of ADC(min) and mean ADC (ADC(mean)) values for differentiating between benign and malignant tumors of the liver. MATERIALS AND METHODS: We retrospectively subjected 240 patients with 195 malignant (hepatocellular carcinoma [HCC], n = 137; metastases, n = 44; cholangiocellular carcinoma [CCC], n = 14) and 45 benign tumors (hemangiomas, n = 37; focal nodular hyperplasia [FNH], n = 8). Both ADC(mean) and ADC(min) were evaluated independently by 2 readers, the sensitivity and specificity for the detection of malignancy were calculated, and receiver operating characteristic (ROC) curves were generated. To determine interobserver agreement, we calculated the Pearson correlation coefficient. RESULTS: Mean ADC (×10 mm/s) was 1.19 for malignant (HCC, 1.15; metastasis, 1.23; CCC, 1.51) and 2.01 for benign tumors (hemangioma, 2.09; FNH, 1.52; P < 0.001). Minimum ADC was 0.81 for malignant (HCC, 0.79; metastasis, 0.81; CCC, 0.91) and 1.62 for benign tumors (hemangioma, 1.66; FNH, 1.28; P < 0.001). The sensitivity, specificity, and the calculated area under the ROC curve for diagnosing malignant lesions were 86.2%, 86.7%, and 0.942 (reader 1) and 88.7%, 88.9%, and 0.939 (reader 2) for ADC(mean); they were of 92.3%, 97.8%, and 0.984 (reader 1) and 94.9%, 97.8%, and 0.983 (reader 2) for ADC(min). CONCLUSIONS: Mean ADC and ADC(min) were valuable for differentiating between malignant and benign hepatic lesions. The area under the ROC curve of ADC(min) was significant higher than that of ADC(mean).

Differences in the incidence of nosocomial-onset COVID-19 among hospitalized patients with exposure to SARS-CoV-2.

We evaluated the secondary COVID-19 incidence among uninfected hospitalized patients after nosocomial COVID-19 exposure. An exposure source of SARS-CoV-2 was hospitalized patients or healthcare personnel (HCP) newly diagnosed as having COVID-19. Patients exposed to a COVID-19-infected patient in a shared room more frequently developed COVID-19 than those exposed to an infected HCP.

A case of pancreatic adenosquamous cell carcinoma with a pseudocyst following curative surgery.

BACKGROUND: Pancreatic adenosquamous cell carcinoma (PASC) is a relatively rare histological type of pancreatic malignancy, and preoperative diagnosis is difficult because of its rarity. PASC accounts for 1-4% of all pancreatic cancers, and even after curative surgery, its prognosis is poorer than that of ordinary pancreatic adenocarcinoma. Pathologically, it shows glandular and squamous differentiation of cells. Complete resection is the only method to achieve a good long-term prognosis, and an increasing doubling time of PASC is considered to indicate early recurrence after surgery. Here, we report a rare case of PASC with an infected pancreatic cyst that was difficult to treat, along with a review of the literature. CASE PRESENTATION: A woman in her 80s with a history of breast cancer presented with pericardial pain. Computed tomography revealed a 20-mm hypovascular tumor in the body of the pancreas and a 27-mm pseudocyst. Endoscopic retrograde cholangiopancreatography showed a severe main pancreatic duct stenosis in the body of the pancreas that made cannulation impossible, and contrast media extravasation was due to pancreatic duct disruption in the pancreatic tail. Endoscopic fine-needle aspiration revealed that the tumor was a PASC. Because the patient had an infected pancreatic cyst, central intravenous nutrition and antibiotics were administered, which stabilized her general condition. She was diagnosed with resectable PASC and underwent distal pancreatectomy with lymphadenectomy. The postoperative course was uneventful. Immunohistochemical analysis of the resected specimen confirmed T2N0M0 stage IB. Systemic adjuvant chemotherapy with S-1 is ongoing. CONCLUSION: Appropriate preoperative management and preoperative accurate staging (T2N0M0 stage IB) of PASC with curative surgery can ensure predictable outcomes.

A case of successful management for spontaneous rupture of paraganglioma treated with preoperative transcatheter arterial embolization.

BACKGROUND: Tumors arising from catecholamine-producing chromophil cells in paraganglia are termed paragangliomas (PGLs), which biologically resemble pheochromocytomas (PCCs) that arise from the adrenal glands. Spontaneous rupture of a PGL is rare and can be fatal. Although elective surgery for ruptured PCCs after transcatheter arterial embolization (TAE) has been shown to provide good outcomes, the efficacy of TAE pretreatment for ruptured PGL remains unknown. CASE PRESENTATION: A 65-year-old female with hypertension and tachycardia was diagnosed with a 3-cm PGL located behind the inferior vena cava. The patient was scheduled to undergo an elective surgery with antihypertensive therapy. However, she presented with a chief complaint of abdominal pain and was diagnosed with intratumoral hemorrhage. Urgent TAE was performed that successfully achieved hemorrhage control. After TAE, serum levels of both epinephrine and norepinephrine were within the normal range. Abdominal computed tomography revealed resolving retroperitoneal hematoma. Elective open surgery was performed without significant intraoperative bleeding or fluctuations in blood pressure. CONCLUSION: We report a case of successful preoperative TAE for functional PGL to control intraoperative blood pressure fluctuations and bleeding. Preoperative TAE could be a useful procedure for the surgical preparation of functional PGL, including unruptured cases.

Evaluation of the short-term complications of intracorporeal anastomosis in right-sided colectomy.

INTRODUCTION: The benefits of intracorporeal anastomosis in laparoscopic colorectal cancer surgery remain unclear. Therefore, we aimed to investigate the short-term postoperative outcomes of intracorporeal anastomosis. METHODS: We retrospectively analyzed 87 patients who underwent laparoscopic surgery for right-sided colon tumors using a colon database. RESULTS: Of the 87 patients, 23 underwent intracorporeal anastomosis and 64 underwent extracorporeal anastomosis. Intraoperative bleeding, wound length, exhaust gas, preoperative white blood cell count, and C-reactive protein (postoperative day 1) were higher in the extracorporeal anastomosis group than in the intracorporeal anastomosis group. The incidence of wound infection was higher in the intracorporeal anastomosis group than in the extracorporeal anastomosis group. In the irrigation water bacterial culture collected after anastomosis, the positive group had a higher white blood cell count on postoperative day 1 and higher C-reactive protein levels on postoperative day 3 than did the negative group. Patients who underwent chemical preparation had lower C-reactive protein levels on postoperative day 1 than did the group who did not undergo chemical preparation. CONCLUSION: Despite the advantages of intracorporeal anastomosis in terms of wound length and intraoperative bleeding, the risk of infection may increase during the introduction phase. Fever and inflammatory responses are significantly elevated in culture-positive cases.

Ferguson plot analysis of multiple intermediate species of thermally unfolded bovine serum albumin.

Ferguson plot was used to characterize the multiple intermediate species of bovine serum albumin (BSA) upon thermal unfolding. Differential scanning calorimetry showed an irreversible melting of BSA in Tris-HCl and phosphate buffers with a mid-transition temperature, Tm, of ∼68 °C. Thermally unfolded BSA was analyzed by agarose native gel electrophoresis stained by Coomassie blue and SYPRO Orange staining as a function of pH or protein concentration. SYPRO Orange was used to stain unfolded proteins. BSA heated at 70 and 80 °C, i.e., above the Tm, formed multiple intermediate species, which depended on the pH between 7.0 and 8.0, protein concentration and which buffer was used. These intermediate species were analyzed by Ferguson plot, which showed that BSA heated at 60 °C had a similar size to the native BSA, indicating that they are either native or native-like state consistent with no SYPRO Orange staining. The intermediate species observed at higher temperatures with the mobility less than that of the native BSA showed a steeper Ferguson plot and were stained by SYPRO Orange, indicating that these species had a larger hydrodynamic size than the native BSA and were unfolded.

Non-Affinity Purification of Antibodies.

Currently, purification of antibodies is mainly carried out using a platform technology composed primarily of Protein A chromatography as a capture step, regardless of the scale. However, Protein A chromatography has a number of drawbacks, which are summarized in this review. As an alternative, we propose a simple small-scale purification protocol without Protein A that uses novel agarose native gel electrophoresis and protein extraction. For large-scale antibody purification, we suggest mixed-mode chromatography that can in part mimic the properties of Protein A resin, focusing on 4-Mercapto-ethyl-pyridine (MEP) column chromatography.

Discontinuation of admission screening for coronavirus disease 2019 (COVID-19) and the impact on in-hospital clusters of COVID-19: Experience at a tertiary-care center.

We evaluated the impact of discontinuing universal preadmission screening for severe acute respiratory coronavirus virus 2 (SARS-CoV-2) on the occurrence of nosocomial clusters of coronavirus disease 2019 (COVID-19) during the SARS-CoV-2 o (omicron) variant period. No increasing trend in nosocomial clusters was observed during community-based surges before and after discontinuation. This finding supports the safety of the practice change.

Performance of Machine Learning Methods Based on Multi-Sequence Textural Parameters Using Magnetic Resonance Imaging and Clinical Information to Differentiate Malignant and Benign Soft Tissue Tumors.

RATIONALE AND OBJECTIVES: To evaluate the performance of a machine learning method to differentiate malignant from benign soft tissue tumors based on textural features on multiparametric magnetic resonance imaging (mpMRI). MATERIALS AND METHODS: We enrolled 163 patients with soft tissue tumors whose diagnosis was pathologically proven (71 malignant, 92 benign). All patients underwent mpMRI. Twelve histographic and textural parameters were assessed on T1-weighted imaging (T1WI), T2-weighted imaging, diffusion-weighted imaging, apparent diffusion coefficient maps, and contrast-enhanced T1WI imaging. We compared mean signals of all sequences from the malignant and benign tumors using Welch's t-test. Prediction models were developed via a machine learning technique (support vector machine) using textural features of each sequence, clinical information (sex + age + tumor size), and the combined model incorporating all features. Areas under the receiver operating characteristic curves (AUCs) of these models were calculated using fivefold cross validation. RESULTS: The diagnostic ability of clinical information model (AUC 0.85) was not inferior to the model with textural features of each sequence (AUC 0.79-0.84). The combined model showed the highest diagnostic ability (AUC 0.89). The AUC of the combined model (0.89) was comparable to those of two board-certified radiologists (0.89 and 0.87). CONCLUSIONS: Machine learning methods based on textural features on mpMRI and clinical information offer adequate diagnostic performance to differentiate between malignant and benign soft tissue tumors.

Analysis of proteins by agarose native gel electrophoresis in the presence of solvent additives.

Solvent additives, including NaCl, arginine hydrochloride (ArgHCl), glycine and sucrose, are used to enhance protein stability or reduce protein aggregation. Here, we studied the effects of these additives on proteins using agarose native gel electrophoresis. Since these additives are used at relatively high concentration, we first confirmed that they do not interfere with the performance of the native gel electrophoresis. Agarose native gel electrophoresis showed that aggregation of bovine serum albumin (BSA) induced by heating was slightly reduced by NaCl and ArgHCl. On the contrary, glycine and sucrose had marginal effects. ArgHCl and NaCl promoted heat aggregation of monoclonal antibody (mAb), while glycine and sucrose stabilized the native mAb. Arginine methyl ester inhibited heat aggregation of lysozyme and, to a much lesser extent, BSA. These results show that agarose native gel electrophoresis can be used to analyze the effects of solvent additives on proteins subjected to heat stresses. SYPRO Orange that stains only unfolded proteins confirmed unfolded structures of soluble aggregates.