Assay Buffer Research Articles

Exercise redox biochemistry: Conceptual, methodological and technical recommendations

Exercise redox biochemistry is of considerable interest owing to its translational value in health and disease. However, unaddressed conceptual, methodological and technical issues complicate attempts to unravel how exercise alters redox homeostasis in health and disease. Conceptual issues relate to misunderstandings that arise when the chemical heterogeneity of redox biology is disregarded: which often complicates attempts to use redox-active compounds and assess redox signalling. Further, that oxidised macromolecule adduct levels reflect formation and repair is seldom considered. Methodological and technical issues relate to the use of out-dated assays and/or inappropriate sample preparation techniques that confound biochemical redox analysis. After considering each of the aforementioned issues, we outline how each issue can be resolved and provide a unifying set of recommendations. We specifically recommend that investigators: consider chemical heterogeneity, use redox-active compounds judiciously, abandon flawed assays, carefully prepare samples and assay buffers, consider repair/metabolism, use multiple biomarkers to assess oxidative damage and redox signalling.

Simultaneous screening for marbofloxacin and ofloxacin residues in animal-derived foods using an indirect competitive immunoassay

ABSTRACTA broad-specificity immunoassay was established to monitor marbofloxacin (MBF) and ofloxacin (OFX) residues in raw milk and porcine muscle samples. The main influential parameters in the assay buffer were investigated to improve the assay performance. The cross-reactivities (CRs) were lower than 0.25%, except for OFX (41.38%). The quantitative working ranges for MBF and OFX based on fortified matrices ranged from 0.11 to 30.85 ng mL−1, with a 50% inhibitory concentration of 0.76±0.12 ng mL−1 for MBF and 2.70±0.28 ng mL−1 for OFX. The detection limits of enzyme-linked immunosorbent assay (ELISA) ranged from 0.053 to 0.358 ng mL−1 in spiked samples. The mean recoveries were in the range of 80.52–95.46%, less than 12.06% of the coefficient of variation. A good correlation between the ELISA results and those from instrument methods demonstrated the practical application of the novel ELISA. Therefore, the immunoassay proposed is feasible for MBF and OFX analysis in animal-derived foods.

Mechanism of formate-nitrite transporters by dielectric shift of substrate acidity.

Bacterial formate-nitrite transporters (FNTs) regulate the metabolic flow of small, weak mono-acids. Recently, the eukaryotic PfFNT was identified as the malaria parasite's lactate transporter and novel drug target. Despite crystal data, central mechanisms of FNT gating and transport remained unclear. Here, we show elucidation of the FNT transport mechanism by single-step substrate protonation involving an invariant lysine in the periplasmic vestibule. Opposing earlier gating hypotheses and electrophysiology reports, quantification of total uptake by radiolabeled substrate indicates a permanently open conformation of the bacterial formate transporter, FocA, irrespective of the pH Site-directed mutagenesis, heavy water effects, mathematical modeling, and simulations of solvation imply a general, proton motive force-driven FNT transport mechanism: Electrostatic attraction of the acid anion into a hydrophobic vestibule decreases substrate acidity and facilitates protonation by the bulk solvent. We define substrate neutralization by proton transfer for transport via a hydrophobic transport path as a general theme of the Amt/Mep/Rh ammonium and formate-nitrite transporters.

P422 Patient-near Infliximab trough-level testing by a novel quantitative rapid test; the Quantum Blue Infliximab assay

Background: Therapeutic drug monitoring (TDM) has become standard clinical practice and overwhelming clinical evidence indicates that dose-optimization improve clinical outcome by decreasing the risk for anti-drug-antibodies (ADA) and improves the efficacy of the drug itself. There is also another aspect for advocating TDM, that is improving the health economic aspect of these very expensive drugs. However, this has been hampered by the high cost of and the absence of a near patient testing. Aims of the study: The study had two aspects; first is to correlate a CE -marked rapid test for IFX trough level, the Quantum Blue Infliximab test (QB-IFX) (BÜHLMANN Laboratories, AG, Basel, Switzerland) to an assay very similar to the Loeven assay. Secondly, to correlate the performance of such a test done by; A) a nurse and B) a trained laboratory person Methods: The study comprised 64 pts with IBD receiving IFX treatment; 14 Remicade and 50 Remsina. At the day of infusion, blood for IFX-trough level was collected in addition to 3 ml serum for QB-IFX rapid test. Part A: A nurse (IL) received one hour of “laboratory” training before running the QB-IFX under supervision of AR. The serum was diluted 10uL in 190 uL assay buffer and vortexed for 5 sec. 70uL was applied to the rapid test cassette and read after 15 min using the Q B reader. Part B: The same procedure was followed by an experienced lab technician (GHM). In addition, 5 aliquots from three sera-levels was collected (3, 7 and 10ug/ml) and tested to establish CV for the upper and lower trough level values as well as for one high level value. Results: The was a very good correlation between the QB-IFX rapid test and the laboratory ELISA test, r=0.90, p<0.001, the slope was 1.1. Furthermore, the correlation between the results obtained by the nurse and a skilled lab person was acceptable with r=0.92, p<0.001. The CV for the upper and lower trough level was 4.7 and 7.8% respectively. CV for the high level was 14.7% Conclusions: This is the first study that documents a close correlation between a 15 min. rapid test for IFX trough level with that of an standard lab-test. We have shown that such a test can accurately be performed by a nurse. The CV for the three different serumlevels was way lower than expected for a lateral flow rapid test. This means that TDM now can be moved from a distant laboratory to the near patient facility like the infusion centre to ensure correct dosing in IBD and other patients on IFX treatment.

Development of a Simple, Fast, and Quantitative Lateral Flow Immunochromatographic Strip for Folic Acid

Folic acid (FA) is an important B vitamin naturally found in foods and also used in dietary supplements and food fortification. To monitor FA content is important to guarantee food quality and also to prevent its abuse in dietary supplements. In this work, we developed a simple and robust lateral flow immunochromatographic assay (ICA) for the routine screening of FA in food samples. FA was conjugated to carrier protein as immunogen and used to produce specific polyclonal antibody (pAb). The pAb was conjugated with gold nanoparticle and used to develop a lateral flow ICA. After the careful optimization of ICA conditions, the assay showed an IC50 of 51.8 ng mL−1 and linear range from 23.4 to 114.5 ng mL−1 for FA in assay buffer, with the assist of a DY6510 strip reader within 8 min. Using milk powder as a matrix model, after a 15-fold dilution to eliminate matrix effect, the ICA showed intra-assay recoveries from 94.2 to 107.0% with coefficient of variance (CV) below 15% and inter-assay recoveries from 91.4% to 111.3% with CV below 18%. Good agreement was obtained between the results of ICA and ciELISA and standard HPLC. It is an ideal screening tool for the fast monitoring of FA in a simple, fast, and low-cost manner.

A novel kindred with inherited STAT2 deficiency and severe viral illness

A novel kindred with inherited STAT2 deficiency and severe viral illness

Measurement of Energy-dependent Rhodamine 6G Efflux in Yeast Species.

Rhodamine 6G is a highly fluorescent dye often used to determine the transport activity of yeast membrane efflux pumps. The ATP-binding cassette transporter KlPdr5p confers resistance to several unrelated drugs in Kluyveromyces lactis. KlPdr5p also extrudes rhodamine 6G (R6G) from intact yeast cells in an energy-dependent manner. Incubation of yeast cells in the presence of 2-deoxy-D-glucose (inhibitor of glycolysis) and R6G (mitochondrial ATPase inhibitor) leads to marked depletion of intracellular ATP pool ( Kolaczkowski et al., 1996 ). An active KlPdr5p mediated extrusion of R6G from intact yeast cells can be followed by direct measurement of the fluorescence of extruded R6G in the assay buffer.

Prim-O-glucosylcimifugin Attenuates Lipopolysaccharideinduced Inflammatory Response in RAW 264.7 Macrophages.

Background:Radix Saposhnikoviae (RS) exerts anti-inflammatory, analgesic, antipyretic, antioxidation effects and has been used in traditional Chinese medicine to treat common colds, headache, and rheumatoid arthritis. Prim-O-glucosylcimifugin (POG) is the highest content chromone and one of the major active constituents in RS.Objective:The study was aimed to explore the anti-inflammation effects of POG in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.Materials and Methods:Cell viability was detected by Cell Counting Kit-8 assay. Production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 was assessed by enzyme-linked immunosorbent assay. Real-time polymerase chain reaction and Western blot were performed to analyze mRNA and protein levels, respectively.Results:During the whole experiment, 15, 50, and 100 μg/mL of POG had no cytotoxicity on RAW 264.7 cells. POG dose-dependently inhibited the production of NO, TNF-α, IL-1β, and IL-6 that were induced by LPS. POG treatment downregulated the mRNA and protein expression inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2) in LPS-activated RAW 264.7 macrophages in a concentration-dependent manner. Furthermore, LPS-induced JAK2/STAT3 activation was prevented in RAW 264.7 macrophages by POG treatment. STAT3 overexpression significantly reversed the effects of POG on LPS-activated RAW 264.7 macrophages.Conclusion:These results demonstrate that POG exerts anti-inflammatory effects through the inhibition of iNOS and COX-2 expression by inhibiting the phosphorylation of JAK2/STAT3.SUMMARY POG exerts anti-inflammatory effects in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 expression by inhibiting JAK2/STAT3 signaling. Abbreviations used: LPS: Lipopolyssacharide; NO: Nitric oxide; TNF-α: Tumor necrosis factor-α; IL: Interleukin; RS: Radix Saposhnikoviae; POG: Prim-O-glucosylcimifugin; iNOS: Inducible NO synthase; COX2: Cyclooxygenase; FBS: Fetal bovine serum; DMSO: Dimethylsulfoxide; CCK-8: Cell Counting Kit; RIPA: Radio immunoprecipitation assay buffer; ECL: Enhanced chemiluminescence; SD: Standard deviation; ELISA: Enzyme-Linked immunosorbent assay.

Development of a modified 3T3 Neutral Red Uptake Phototoxicity Test protocol for evaluation of poorly water-soluble substances.

The 3T3 neutral red uptake phototoxicity test (OECD TG432) is an alternative phototoxicity test method that is relatively easy and rapid to implement, with results obtainable in a short time, and is reported to have high reproducibility compared with in vivo assay methods. However, this method has been shown to be unsuitable for testing poorly water-soluble substances, which tend to separate out when mixed with the assay buffer solution. This causes difficulties in determining the dose dependency of substances and subsequent determination of the photoirritation factor because the ratio of cell viability, expressed as the half-maximal inhibitory concentration (IC50) in the presence or absence of light, is not calculable. In this study, we investigated the optimum conditions for the evaluation of poorly water-soluble substances. In the conventional method, the final solvent concentration was 1% and the pre-incubation time was 60 min, but in the modified method, 10% and 5 min were used, respectively. Next, the results from the conventional method were compared with those of our modified method, which was found to be viable and comparable with the conventional method. Moreover, the false positive results frequently obtained with poorly water-soluble substances in the conventional method were not evident with the modified method, thus confirming its usefulness for the evaluation of such substances. We therefore propose that the modified method can be used for the in vitro testing of poorly water-soluble substances in phototoxicity evaluations.

Comparability of the effect of storage time and temperature on serum anti-Müllerian hormone measurement between original and modified enzyme-linked immunosorbent assay

ObjectiveTo explore the effect of modified enzyme-linked immunosorbent assay on the AMH results is increased or decreased, and to investigate the effect of storage time and temperature on AMH measurements with and without sample premixing assay buffer using the Kangrun ELISA method. MethodSerum AMH concentration were measured by ELISA, consistency between two kits, and comparability between original and the modified assay under different stored conditions were analyzed by Passing-Bablok regression analysis and Bland-Altman bias evaluation. ResultThere was a strong consistency between AMH concentrations measured in Kangrun ELISA and Ansh Labs ultra-sensitive AMH ELISA. Pre-mixing serum specimens with assay buffer gave consistent results compared with original assay. Modified protocol can reduce the amplitude of increase affected by sample aged and give the most consistent results regardless of storage conditions. ConclusionPre-mixing protocol did not influence the results of fresh serum or frozen serum incubation <3days at 4°C and −80°C, but when specimens detected after collection and stored in other storage conditions, should be pre-mixed with assay buffer to insure its accuracy.

Radioimmunoassay of relaxin-like gonad-stimulating peptide in the starfish Patiria (=Asterina) pectinifera

A relaxin-like gonad-stimulating peptide (RGP) from starfish Patiria (=Asterina) pectinifera is the first identified invertebrate gonadotropin for final gamete maturation. An antiserum against P. pectinifera RGP (PpeRGP) was produced by immunizing rabbits with a PpeRGP sulfanyl-polyethylene glycol derivative conjugated with keyhole limpet hemocyanin (KLH) as the antigen. The antiserum was used for the development of a specific and sensitive radioimmunoassay (RIA) for the measurement of RGP. In binding experiments using radioiodinated PpeRGP and antiserum against PpeRGP, a displacement curve was obtained using radioinert PpeRGP. The sensitivity of the RIA, defined as the amount of PpeRGP that significantly decreased the counts by 2 SD from the 100% bound point, averaged 0.040±0.002pmol PpeRGP per 100μl assay buffer (0.40±0.02nM) in 10 assays. Intra-assay and inter-assay coefficients of variation were 6.1% and 2.7%, respectively. Serial dilution of whole homogenates from the radial nerve cords and circumoral nerve-rings of P. pectinifera produced displacement curves parallel to the PpeRGP standard. Thus, the amounts of PpeRGP were determined as 1.54±0.09pmol/mg wet weight of radial nerves and 0.87±0.04pmol/mg wet weight of nerve-rings, respectively. On contrary, pyloric stomach, pyloric caeca, tube-feet, ovaries, testes, and ovarian follicle cells did not react in the RIA system. Furthermore, the A- and B-chains of PpeRGP, Asterias amurensis RGP, bovine insulin, and human relaxin did not show cross-reactivity in the RIA. These results strongly suggest that the RIA system is a highly specific and sensitive with respect to PpeRGP.

Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes

Sphingosine-1-phosphate (S1P) is present in the blood plasma and acts as a pivotal intercellular signal transmitter in the immune system by recruiting lymphocytes from the thymus and secondary lymphoid tissues. The plasma S1P concentration is maintained by the supply of S1P from erythrocytes. Previously, we showed that S1P release from erythrocytes is mediated by an ATP-dependent transporter. In this study, we attempted to establish a rapid and reliable method for measuring the S1P transport activity in erythrocytes by using a fluorescent S1P analog, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled S1P. NBD-S1P was released from erythrocytes in a time-dependent manner. The NBD-S1P release was reduced after exposure to glyburide, which is an inhibitor of the S1P transporter in erythrocytes. Moreover, the release of NBD-S1P and S1P from erythrocytes was competitively inhibited by intracellular S1P and NBD-S1P, respectively. These results showed that the erythrocyte S1P transporter exports NBD-S1P. We optimized the sample-preparation conditions and lipid extraction to increase the sensitivity of the assay. Furthermore, we successfully measured NBD-S1P release without lipid extraction by decreasing the concentration of BSA in the assay buffer to 0.1%. This method will be useful for the high-throughput screening of S1P transporter inhibitors using conventional fluorometers.

Fast simultaneous detection of three pesticides by a White Light Reflectance Spectroscopy sensing platform

The simultaneous determination of pesticides chlorpyrifos, imazalil and thiabendazole in drinking water and wine samples with a biosensor based on White Light Reflectance Spectroscopy is presented. A competitive immunoassay format was implemented followed by reaction of immunoadsorbed anti-analyte specific antibody molecules with a secondary antibody in order to enhance the signal and reduce the analysis time. The whole analysis was completed in less than 10min and the limits of quantification achieved with calibrators prepared in buffer were 60pg/mL for chlorpyrifos and imazalil, and 80pg/mL for thiabendazole. Bottled water samples were analysed without any treatment, whereas white and red wine samples were analysed after 10-times dilution with assay buffer. This way pesticide concentrations as low as 0.6ng/mL for chlorpyrifos and imazalil, and 0.8ng/mL for thiabendazole could be detected in wines. The measurements were repeatable with intra- and inter-assay CVs values ranging from 4.9 to 8.2% and from 7.3 to 9.7%, respectively, for all assays. The accuracy of the measurements was evaluated through recovery experiments using spiked water and wine samples. Percent recovery values ranged from 86 to 116%, demonstrating the accuracy of the measurements performed with the developed biosensor. In addition, 5 wines spiked with different concentrations of the three pesticides were analysed both with the developed biosensor and a validated LC–MS/MS method. The good agreement of the results obtained with the two methods is a further testimony of the developed method accuracy and a good starting point to demonstrate its Point-of-Need application capabilities in the food analysis field.

Does a voltage-sensitive outer envelope transport mechanism contributes to the chloroplast iron uptake?

Based on the effects of inorganic salts on chloroplast Fe uptake, the presence of a voltage-dependent step is proposed to play a role in Fe uptake through the outer envelope. Although iron (Fe) plays a crucial role in chloroplast physiology, only few pieces of information are available on the mechanisms of chloroplast Fe acquisition. Here, the effect of inorganic salts on the Fe uptake of intact chloroplasts was tested, assessing Fe and transition metal uptake using bathophenantroline-based spectrophotometric detection and plasma emission-coupled mass spectrometry, respectively. The microenvironment of Fe was studied by Mössbauer spectroscopy. Transition metal cations (Cd2+, Zn2+, and Mn2+) enhanced, whereas oxoanions (NO3-, SO42-, and BO33-) reduced the chloroplast Fe uptake. The effect was insensitive to diuron (DCMU), an inhibitor of chloroplast inner envelope-associated Fe uptake. The inorganic salts affected neither Fe forms in the uptake assay buffer nor those incorporated into the chloroplasts. The significantly lower Zn and Mn uptake compared to that of Fe indicates that different mechanisms/transporters are involved in their acquisition. The enhancing effect of transition metals on chloroplast Fe uptake is likely related to outer envelope-associated processes, since divalent metal cations are known to inhibit Fe2+ transport across the inner envelope. Thus, a voltage-dependent step is proposed to play a role in Fe uptake through the chloroplast outer envelope on the basis of the contrasting effects of transition metal cations and oxoaninons.

Abstract LB-043: Improving rectal cancer therapy using a patient-derived tumor xenograft model

Abstract Mechanisms of locally advanced rectal cancer (LARC) neoadjuvant chemoradiation resistance are poorly understood. Current research models to study this issue are incomplete and restricted by the limited supply of untreated tissue samples. Patient-derived xenografts (PDX) are commonly developed from surgical specimens and can serve as effective tools to investigate therapy. We previously demonstrated that the SNAI2/ miR-145 signaling axis enhanced the tumor-initiating cell (TIC) and chemotherapy resistance. We developed a unique PDX model from pre-neoadjuvant therapy LARC biopsy samples to study chemoradiation resistance. Further, we hypothesized that TICs isolated from LARC patient-derived xenografts would demonstrated increased SNAI2/ miR-145 signaling. Methods: Fresh LARC endoscopic biopsy samples were digested using Liberase DH to yield a dissociated single-cell solution. All in vivo experiments consisted of injecting cells with Matrigel subcutaneously into NSG NODSCID/IL2Rgnull immunocompromised mice. Xenograft (Passage 1, P1) single cells were resuspended in ALDEFLUOR assay buffer. ALDEFLUOR and EPCAM positive cells (ALDH+/EPCAM+) were then detected using a flow cytometer to identify the TICs. Using the negative control ALDH inhibitor, DEAB (1.5 μM), identify the ALDH+ cells compared with background. For limited dilution (LD) in vitro spheroid assay, 5000 EPCAM+/ALDH+ and ALDH- cells were cultured in ultra-low attachment plates in cancer stem-cell medium (DMEM-F12; 1X B-27, βEGF, βFGF, 1% penicillin/streptomycin, BSA, heparin, β-mercaptoethanol). For LD in vivo study, EPCAM+ ALDH sorted cells were implanted subcutaneously with Matrigel and closely observed. Droplet Digital PCR was conducted using the QX200 Droplet Digital PCR System. miR-145 PCR was performed using TaqMan MicroRNA Assays (Applied Biosystems) and normalized to RNU6B. Results: Our LARC PDX model had high outgrowth and engraftment rates of 70 and 85% respectively and maintained critical histologic features. Both LD in vitro and in vivo experiments demonstrated that EPCAM+/ALDH+ cells were highly tumorigenic suggestive of TICs (p&amp;lt;0.05). The ALDH+ population enriched for SNAI2/ miR-145 signaling. miR-145 was inhibited by at least 8.8 fold in the TIC population. Conclusions: The lack of novel strategies plays a central role in the inability to overcome therapeutic resistance in LARC. Our results demonstrate a novel model to investigate LARC and suggest that a novel miR-145 therapeutic strategy may be able to target the TIC in LARC patients. Citation Format: Ernest R. Camp, Cindy Wang, Katie Hurst, Lourdes M. Nogueira, Victoria J. Findlay. Improving rectal cancer therapy using a patient-derived tumor xenograft model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-043.

Examining the Effects of Sodium Ions on the Binding of Antagonists to Dopamine D2 and D3 Receptors.

Many G protein-coupled receptors have been shown to be sensitive to the presence of sodium ions (Na+). Using radioligand competition binding assays, we have examined and compared the effects of sodium ions on the binding affinities of a number of structurally diverse ligands at human dopamine D2 and dopamine D3 receptor subtypes, which are important therapeutic targets for the treatment of psychotic disorders. At both receptors, the binding affinities of the antagonists/inverse agonists SB-277011-A, L,741,626, GR 103691 and U 99194 were higher in the presence of sodium ions compared to those measured in the presence of the organic cation, N-methyl-D-glucamine, used to control for ionic strength. Conversely, the affinities of spiperone and (+)-butaclamol were unaffected by the presence of sodium ions. Interestingly, the binding of the antagonist/inverse agonist clozapine was affected by changes in ionic strength of the buffer used rather than the presence of specific cations. Similar sensitivities to sodium ions were seen at both receptors, suggesting parallel effects of sodium ion interactions on receptor conformation. However, no clear correlation between ligand characteristics, such as subtype selectivity, and sodium ion sensitivity were observed. Therefore, the properties which determine this sensitivity remain unclear. However these findings do highlight the importance of careful consideration of assay buffer composition for in vitro assays and when comparing data from different studies, and may indicate a further level of control for ligand binding in vivo.

Cardiomyocyte-Specific Human Bcl2-Associated Anthanogene 3 P209L Expression Induces Mitochondrial Fragmentation, Bcl2-Associated Anthanogene 3 Haploinsufficiency, and Activates p38 Signaling

The Bcl2-associated anthanogene (BAG) 3 protein is a member of the BAG family of cochaperones, which supports multiple critical cellular processes, including critical structural roles supporting desmin and interactions with heat shock proteins and ubiquitin ligases intimately involved in protein quality control. The missense mutation P209L in exon 3 results in a primarily cardiac phenotype leading to skeletal muscle and cardiac complications. At least 10 other Bag3 mutations have been reported, nine resulting in a dilated cardiomyopathy for which no specific therapy is available. We generated αMHC-human Bag3 P209L transgenic mice and characterized the progressive cardiac phenotype invivo to investigate its utility in modeling human disease, understand the underlying molecular mechanisms, and identify potential therapeutic targets. We identified a progressive heart failure by echocardiography and Doppler analysis and the presence of pre-amyloid oligomers at 1 year. Paralleling the pathogenesis of neurodegenerative diseases (eg, Parkinson disease), pre-amyloid oligomers-associated alterations in cardiac mitochondrial dynamics, haploinsufficiency of wild-type BAG3, and activation of p38 signaling were identified. Unexpectedly, increased numbers of activated cardiac fibroblasts were identified in Bag3 P209L Tg+ hearts without increased fibrosis. Together, these findings point to a previously undescribed therapeutic target that may have application to mutation-induced myofibrillar myopathies as well as other common causes of heart failure that commonly harbor misfolded proteins.

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors.

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping. Tissue specimens are harvested and stored at -80 °C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in (125)I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: 'non-specific binding' (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and 'total binding' with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 µM) is also present in the incubation buffer to limit (125)I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 °C, NSP slides are exposed to 10 µM PD123319 and losartan, while 'total binding' slides are exposed to 10 µM PD123319. Slides are then incubated with (125)I-SI-Ang II in the presence of PD123319 for 'total binding', and PD123319 and losartan for NSP in assay buffer, followed by several 'washes' in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons. The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Assays for Determination of Protein Concentration.

Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also included is a rapid, inexpensive, and reliable BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels. © 2016 by John Wiley & Sons, Inc.

Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System.

Baculovirus-Bombyx mori protein expression system has mainly been used for translation of eukaryotic proteins. In contrast, information pertaining to bacterial protein expression using this system is not sufficient. Therefore, recombinant nucleases from Serratia liquefaciens (rSlNucAs) were expressed in a Baculovirus-B. mori protein expression system. rSlNucAs containing the native signal peptide (rSlNucA-NSP) or silkworm 30-K signal peptide (rSlNucA-30K) at the NH2-terminus were constructed to enable secretion into the extracellular fraction. Both rSlNucA-30K and rSlNucA-NSP were successfully secreted into hemolymph of B. mori larvae. Affinity-purified rSlNucAs showed high nuclease activity. Optimum pH was 7.5 and half of maximum activity was maintained between pH 7.0 and 9.5. Optimum temperature was 35°C. rSlNucAs showed sufficient activity in twofold-diluted radioimmunoprecipitation assay buffer and undiluted, mild lysis buffer. Genomic DNA of Escherichia coli was efficiently digested by rSlNucAs in the bacterial lysate. The results in this study suggest that rSlNucAs expressed by the Baculovirus-B. mori protein expression system will be a useful tool in molecular biology. Functional recombinant protein of bacteria was produced by Baculovirus-B. mori protein expression system. This system may be highly suitable for bacterial extracellular protein secreted via Sec pathway.