Protein Assay Method Research Articles

Integrated CRISPR/Cas12a with terminal protection strategy for homogeneous protein assay

Developing appropriate protein assay methods is necessary, as protein assays play an important role in molecular diagnosis, drug screening, and biomedical research. This article presents a universal homogeneous method based on the CRISPR/Cas12a system and the terminal protection strategy for protein assays. Initially, a single-stranded DNA (ssDNA) labeled with a small molecule at its 3’-end was designed to respond to the target protein and serve as an activator of Cas12a. In the absence of the target protein, small-molecule-linked ssDNA was effectively cleaved using exonuclease I (Exo I). The lack of the activator resulted in the trans-cleavage inactivity of Cas12a, leading to low fluorescence in the detection solution. However, in the presence of the target protein, the specific binding of the target protein to the small molecules on the activator protected the activator against digestion by Exo I. The remaining intact activator triggered the trans-cleavage activation of Cas12a, producing strong fluorescence. Based on this principle, our method achieved fluorescence enhanced detection of the target protein. After experimental condition optimization, our method achieved highly sensitive and specific protein detection in two models: folate receptor and streptavidin. The excellent performance of the fluorescence method highlights the applicability and effectiveness of the CRISPR/Cas12a system in protein assays, and broadening its application scope.

Difference in urinary protein-to-creatinine ratio using the benzethonium chloride and pyrogallol red methods in children.

The appropriate reference value of the urinary protein-to-creatinine ratio (PCR) for proteinuria may change when the urinary pyrogallol red (PR) protein assay method is changed to the benzethonium chloride method (BC). This study aimed to evaluate the difference between BC-based PCR (BC-PCR) and PR-based PCR (PR-PCR) values in children. We compared the BC-PCR and PR-PCR values in the same first-morning urine samples without significant proteinuria in school urine screening settings. The upper limit of the reference values was set at the 97.5th percentile. Notably, 133 samples from 124 individuals (female: 62%, age: median 12.3 years, range 6.3-16.9 years) were collected between August 2020 and October 2022. The diagnoses included 34 normal individuals and 99 with asymptomatic hematuria. The urinary protein (UP) concentrations measured using the BC (BC-UP) and PR (PR-UP) methods were in a linear relationship; however, the BC-UP concentrations were higher than the PR-UP concentrations (mean of differences: 11.2, 95% confidence interval (CI): 11.0-13.4 mg/dL). Also, the BC-PCR values were higher than the PR-PCR values (mean of differences: 0.090, 95% CI: 0.082-0.098 g/gCr). The BC-PCR showed a body-size-related decrease, reflecting a body-size-related urinary creatinine increase. The suggested BC-PCR reference values for proteinuria were 0.25 and 0.17 g/gCr for elementary (6-12.4 years) and junior high school students (12.5-16 years), respectively. These values were higher than those of the PR-PCR and need further studies. When evaluating PCR results, the urinary protein assay should be stated.

Improving Protein Assay Methods to More Accurately Assess Medical Device Cleanliness.

Protein assays commonly used to evaluate reusable device cleanliness do not always accurately measure the low concentrations of protein that are expected on reusable medical devices after processing. Methods often are adapted to provide an estimation of protein concentration; however, sensitivity issues in the portion of standard curves at the acceptance criteria of 6.4 μg/cm2 protein have been reported. Using analytical validation criteria, method improvements for the micro-bicinchoninic acid assay for protein residuals are demonstrated by incorporating a standard addition method, increasing the well volume, and changing the working reagent ratio. These improvements increased method sensitivity and accuracy in the reliable detection of protein levels for device cleaning validations.

Sensitive and rapid Bradford and BCA protein assay by a common desktop scanner

Introduction and Aim: The conventional method of protein quantification involves the need for a costly spectrophotometer which is a bulky and expensive instrument. Also, it requires a considerable quantity of protein which is often valuable or not affordable. In this communication, we report a sensitive image-based protein assay method overcoming the challenges faced by the conventional method. BCA and Bradford protein assay in a miniaturized form is carried out on a polypropylene micro-test plate (PPµTP) using only 1 µl of the precious protein solution in the nanogram range without the need for expensive equipment for quantification. Materials and Methods: In this procedure, after the assay reaction, the assay plate with a color solution is turned upside down for capturing images in a desktop scanner. The image is then quantified digitally by a color space using freely available Adobe Photoshop and Macbeth color calculator software. Results: Standard graphs made by the present image–based method agreeably correlate with the absorbance–based method carried out in a microtiter plate with a Pearson coefficient of 0.995178 and 0.981006 for BCA and Bradford assay respectively. A test protein quantified by this method shows an accuracy of 97%. Further, we have reduced the protein assay time to 75 s only by performing the assay on PPµTP by microwave irradiation. Conclusion: Image-based protein assay is also performed in an ultra-miniature assay plate (UAP) which requires only 1 µl of assay solution, reducing the detection limit further to the 10 femtogram/test zone. In short, image–based protein assay on PPµTP and UAP platforms could be an outstanding alternative either to spectrophotometric or paper–based protein assay.

Opto-Microfluidic Integration of the Bradford Protein Assay in Lithium Niobate Lab-on-a-Chip

This paper deals with the quantification of proteins by implementing the Bradford protein assay method in a portable opto-microfluidic platform for protein concentrations lower than 1.4 mg/mL. Absorbance is measured by way of optical waveguides integrated to a cross-junction microfluidic circuit on a single lithium niobate substrate. A new protocol is proposed to perform the protein quantification based on the high correlation of the light absorbance at 595 nm, as commonly used in the Bradford method, with the one achieved at 633 nm with a cheap commercially available diode laser. This protocol demonstrates the possibility to quantify proteins by using nL volumes, 1000 times less than the standard technique such as paper-analytical devices. Moreover, it shows a limit of quantification of at least 0.12 mg/mL, which is four times lower than the last literature, as well as a better accuracy (98%). The protein quantification is obtained either by using one single microfluidic droplet as well by performing statistical analysis over ensembles of several thousands of droplets in less than 1 min. The proposed methodology presents the further advantage that the protein solutions can be reused for other investigations and the same pertains to the opto-microfluidic platform.

Sensitive and rapid determination of heat shock protein 70 using lateral flow immunostrips and upconversion nanoparticle fluorescence probes.

Heat shock protein 70 (Hsp70), belonging to the heat shock protein (HSP) family, is reported to be a potential diagnostic biomarker. In this work, a lateral flow immunostrip was fabricated for the sensitive and rapid determination of Hsp70 by the incorporation of fluorescence and upconversion nanoparticle probes. The upconversion nanoparticles (UCNPs, size ∼39 nm, λex = 980 nm; λem = 540 nm) consisting of a NaYF4:Yb/Er core and polyacrylic acid-modified shell were covalently coupled with Hsp70 antibodies to form the signal probe, which was characterized by dynamic light scattering and zeta potential analyses. The lateral flow assay (LFA) was constructed based on the sandwich-type immunoassay using a sample pad, a test pad, and an adsorption pad on a PVP backing. Hsp70 antibody, IgG antibody and the signal probe were separately dropped on the test zone, the control zone of the test pad, and the sample pad, respectively. In the sandwich LFA, since two antibodies bind to Hsp70 antigenic epitopes, i.e. specific binding, it provided superior specificity and high sensitivity, making it an ideal sensing platform for complex samples like serum Hsp70 samples. The important parameters for the preparation of the lateral flow immunostrips were optimized. Under the optimized conditions, Hsp70 can be detected using the increased fluorescence intensity of UCNPs with a wide linear range from 0.11 to 12 ng mL-1, low detection limit of 0.06 ng mL-1, small sample volume (120 μL), short assay time (15 min) and good reproducibility. The fluorescence method was successfully applied in the determination of Hsp70 in serum samples with good recovery. By combining the accessibility of the lateral flow immunostrips and upconversion nanoparticles, the fluorescence method can serve as a point-of-care testing method for protein assays with high sensitivity and fast detection.

Assessment of lectinic activity potentials in extracts of some tropical Euphorbiaceace

Lectins bind a variety of cells having cell surface glycoprotein or glycolipid, such as erythrocytes, leukemic cells, yeast and several types of bacteria. Several specificity groups have been identified such as mannose, galactose, N-acetyl glucoseamine, N-acetyl galactoseamine, L-fucose and N-acetyl neuraminic acid. The presence of two or more binding sites for each Lectin molecule allows the agglutination of many cell type. Sixteen (16) species of some tropical Euphorbiaceae plants were assessed for the presence of Lectins. The leaves of Acalypha torta, Acalypha wiskesiana, Acalypha hispida, Codiaeum variegatum, Euphorbia milli, Euphorbia pulcherima, Jatropha curcas and Jatropha gossypifola; the seeds of Croton tiglium, Ricinus comminus and Tetracarpidium conophorum; the stem of Euphorbia tirucalli and the tubers of Manihot esculenta (Cassava, vitamin A variety), Manihot esculenta (cassava, NR 8082 variety), Manihot esculenta (Cassava, TMX 419 variety), Manihot esculenta (Cassava, TMX 4(2) 1425 variety) were used for sourcing the Lectins. A. torta, A. wiskesiana, C. variegatum, C. tiglium, E. milli, E. pulcherima, E. tirucalli, J. curcas, J. gossypifola, R. comminus and T. conophorum agglutinated pooled washed human ABO cells in saline (direct haemagglutination) while A. hispida and the four varieties of M. esculenta showed no agglutination reaction. E. pulcherima showed specificity for B cells only while E. tirucalli showed specificity for O cells only, hence could be rightly indicated by referring to them as anti-B Ep and anti-H Et Lectins respectively (where Ep=Euphorbia pulcherima and Et= Euphorbia tirucalli). However A. torta and T. conophorum cross-reacted with pooled washed ABO cells in differing strengths and when standardized, showed that A. torta at a titre of 16 reacted specifically with O cells and T. conophorum at a titre of 128 reacted specifically with B cells. Based on this, these Lectins could be indicated as anti-H At and anti-B Tc respectively (Where At= Acalypha torta and Tc= Tetracarpidium conophorum). The protein content of the crude extracts of the sixteen (16) species were also assayed using Biuret protein assay method and the results revealed that there are no relation or association between the quantity of protein content and agglutination patterns of the extracts. This research has therefore succeeded in revealing presence of Lectinic properties in extracts of some tropical Euphorbiaceae.

Milk quality improvement through the application of plantaricin IIA-1A5 as biopreservatives on fresh milk in dairy cow farm

Foods are better being consumed raw and fresh, however, fresh milk has different case. In fact, drinking fresh milk still became a debate until this day. Some claims said unprocessed fresh milk contains more nutrition compared with pasteurized milk, although milking process do not guarantee protection from bacteria and dirt contamination. One type of bacteriocin known is plantaricin IIA-1A5, this bacteriocin has role as natural biopreservative because it contains antimicrobial compound which expected to be able to damage and kill pathogenic bacteria such as S. aureus by inhibiting other bacteria growth with damaging its cell membranes. This study data analysis was conducted using randomized block design (RDB) with 3 repetitions. Treatment design consisted of control, plantaricin (11.02 mL), and penicillin (7.5 mL). Microbiological characteristics, physicochemical characteristics, and Lowry Protein Assay Method tests were carried out. The results showed that microbiological characteristics and physicochemical characteristics have met SNI Standards, namely 01-3951:1995, 7388:2009, and 3141.1:2011.

Evaluation of Heating and Irradiation Methods for Production of Purified Protein Derivative (PPD) of Mycobacterium Tuberculosis

Tuberculin skin test, also known as the tuberculin or purified protein derivative (PPD) test, is an extensively applied diagnostic test for the detection of primary infection with Mycobacterium tuberculosis (Mtb). The production of PPD is accompanied by some difficulties that require a series of modifications in the production and purification processes. The present study aimed to determine the facilitation level of the manufacturing process by modifying evaluation methods for the production of PPD tuberculin. Mtb strains were cultured in Lowenstein-Jensen media, and the cultured strains were inoculated into the Dorset-Henley liquid medium by the biphasic medium of potato-Dorset-Henley. After incubation, flasks containing cultured strain were selected for bacterial inactivation, and the optimal gamma radiation dose(s) was determined. Tuberculoproteins were precipitated by ammonium sulfate (AS) and Trichloroacetic acid (TCA). Protein concentration was determined using the Bradford and Kjeldahl protein assay methods. Finally, the lymphocyte transformation test and potency test were performed. Based on the results, the Dorset-Henley liquid medium is suitable for the massive growth of the bacterium. The transferal of Mtb from solid to liquid medium was directly carried out without intermediate culture. It was found that during tuberculoprotein production, heating at 100°C for 3 h would be safe for killing mycobacterium. Furthermore, the simultaneous use of heating and gamma irradiation (8 kGgy) killed all of the mycobacteria, while doses of 1, 1.5, and 7 kGy decreased a significant number of bacterial cells. The results also indicated that the concentration of tuberculoprotein extracted by TCA precipitation method was higher than that obtained by AS precipitation. The tuberculoproteins which were produced by these two methods in the lymphocyte transformation test were not significantly different in terms of potency (P>0.05). Moreover, due to the high volume of produced protein, the protein measurement was more efficiently carried out by the Kjeldahl method, compared to the Bradford method. Finally, the results of the present study demonstrated that in addition to the novel approach of gamma irradiation, optimum methods are efficient and applicable in the production of PPD tuberculin.

PENENTUAN KADAR PROTEIN PADA SUSU KEDELAI (Glycinemax L. Merril) DENGAN MENGGUNAKAN METODE Kjeldahl

Soy milk has a combination of nutrients that are almost the same as cow's milk, but soy milk has low cholesterol, gluten, and lactose levels so that soy milk is safe for consumption by people with lactose intolerance, vegetarians, and is also safe for health. Determination of protein content that is most often done is the determination of crude protein which aims to determine the total amount of protein in food ingredients. The most commonly used protein assay method is the Kjeldahlmethod. This study aims to determine the protein content of soy milk packaged in boxes that are made by themselves and sold in traditional markets. The samples used in this study used packaged soy milk, traditional soy milk, and homemade soy milk. The results showed that the protein content of the three samples determined by the Kjeldahlmethod was 3.35±0.0816% traditional soy milk, 2.31±0.3562% box-packed soy milk, and 2.62±0.2495% whole milk. Homemade soybeans. The protein content in box-packed soy milk, homemade soy milk, and traditional soy milk haveeach met the requirements of SNI (1995). The results showed that the protein content of traditional soy milk was greater than that of packaged soy milk and homemade soy milk.

Development of an o-pthalaldehyde (OPA) assay to measure protein content in Ricin Vaccine E. coli (RVEc™)

A recombinant ricin vaccine from E. coli (RVEc™), was developed at the US Army Medical Research Institute of Infectious Diseases (USAMRIID) and assessed in an FDA sponsored Phase 1a clinical trial. At the maximum dosage, two of the study participants developed physiological responses that were elevated to the level of severe adverse reactions. To stay within safe dosing guidelines, the FDA recommended that an assay be developed to accurately quantify the recombinant protein content in the vaccine. The RVEc™ vaccine Final Drug Product (FDP) contains the adjuvant Alhydrogel®, which by its colloidal nature interferes with most conventional protein assay methods. We decided to develop an assay measuring RVEc™ FDP using o-pthalaldehyde (OPA) reagent. The OPA reagent reacts to the primary amines and lysine side chains of proteins in the presence of a thiol under alkaline conditions with a quantifiable fluorescent signature, but does not react with Alhydrogel®. Protein content in the RVEc™ FDP can be determined by comparing the fluorescence of the test sample to the fluorescence of a standard curve of defined concentration. Each phase of the assay was tested to optimize and simplify the assay procedure. The accuracy, specificity, reproducibility, and stability of the assay were evaluated. Results indicated that the optimized and modified OPA assay was simple and able to quantify antigen concentration from a standard curve in the 25 µg/mL–600 µg/mL range. The assay accuracy and coefficient of variation (CV) was 95% and less than 8%, respectively, when determining the ricin protein content in the 200 µg/mL vialed RVEc™ FDP. The assay was simple to perform and used conventional laboratory equipment. This assay could be adapted to measure the protein content in the FDP of other vaccines, but with the proviso that each step of the assay would need to be optimized for each antigen.

Selection of appropriate protein assay method for a paper microfluidics platform

BackgroundPaper-analytical devices (PADs) have gained popularity as a simple and low-cost alternative for determining a wide range of analytes including proteins. Even though several colorimetric PADs methods for protein estimation are reported in literature, they lack justification for the chosen method and parameters therein. AimMajor aim of this work was to thoroughly evaluate the most commonly used colorimetric protein assays and recommend the most appropriate method for PADs platform. MethodWe performed following six colorimetric protein assays on PADs: biuret, lowry, bicinchoninic acid, bradford, bromocresol green, and tetrabromophenol blue. We obtained assay signal by analyzing images of the PADs and then assessed analytical figures of merit. ResultPrecision, accuracy, LOD, and LOQ of PADs protein assay methods ranged from 1.2 to 6.4%, 73.3–102.4%, 0.3–3.8 ​mg/mL, and 1.2–12.8 ​mg/mL, respectively. Out of six methods, we determined bromocresol green and tetrabromophenol blue as the best methods for serum and urine samples, respectively based on their optimized parameters and analytical figures of merit. The total average serum and urine protein in human samples were found to be 94.6 ​± ​16.2 ​mg/mL and 2.1 ​± ​1.5 ​mg/mL, respectively using PADs methods. The PADs result on human samples moderately correlated with the results from spectrophotometric determination (r2 ​> ​0.6). ConclusionPaper-based protein assays were easy to perform and were completed with thousand-fold less volume of samples/reagents without a spectrophotometer compared to conventional assay methods. After testing human samples, we found one protein assay method may not be appropriate for all types of samples.

In Vivo Antioxidant Potential of Protein Hydrolysates of some Cucurbitaceae Seed

Nowadays, dietary proteins play a very crucial role against oxidation, which is a fundamental process in the occurrence of many diseases. The aim of this study was to hydrolyze globulin fractions from C. moschata (CMH), C. lanatus (CLH) and L. siceraria (LSH), and to evaluate their in vivo antioxidant potential. The in vivo antioxidant potential was conducted using in vivo catalase and lipid peroxidation (LPO) assay methods. The total protein content was also estimated using Bio-Rad protein assay and bovine serum albumin methods. For in vivo study, ethanol was induced into test animals for toxicity and oxidative damage for 15 days. The catalase and LOP of serum / liver homogenate were determined. The results showed that globulin hydrolysates decrease the MDA level and increase the catalase level in normal mice. Based on these findings, this study provides information about in vivo antioxidant activities of C. moschata, C. lanatus and L. siceraria hydrolysates. Globulin hydrolysates of L. siceraria showed effective antioxidant properties in vivo models, whereas globulin hydrolysates of C. moschata and C. lanatus exhibited remarkable antioxidant properties, as well. Thus, the results suggest that globulin hydrolysates might be used as a novel source in reducing the risk of oxidative stressed diseases and development of functional foods.
 Keywords: cucurbitaceae, globulin, antioxidant, trypsin

335P - PD-L1 profile of nasopharyngeal cancer patients in Indonesia

BackgroundMolecular and biological characterization of nasopharyngeal cancer (NPC) is scarce. Clinical trials of emerging treatment such as immunotherapy for NPC in endemic area is lacking. Currently the most extensively studied immune related molecule that potentially become the target for treatment is PD-1/PD-L1. PD-L1 profile of NPC patients in Indonesia has never been studied before. This study aimed to elucidate PD-L1 profile from NPC specimens in Indonesia. MethodsNasopharyngeal biopsy specimen was taken for PD-L1 protein examination with enzyme-linked immunosorbent assay (ELISA) method. Tissue specimen was homogenized and then ELISA test was conducted using ABCAM AB214565 kit. All protocols from the manufacturer kit was followed. Total protein concentration from homogenized tissue specimen was obtained through Bradford protein assay method. Then, PD-L1 concentration per mg protein of tissue specimen was calculated. ResultsTwenty-four nasopharyngeal biopsy specimens was obtained. The average age of all patients was 45 years old. Sixteen (66.67%) out of 24 specimens were confirmed histologically to be undifferentiated nasopharyngeal carcinoma. The rest, 8 other specimens have normal histology. Average PD-L1 concentration from NPC specimens was 2,186.67 ± 1,399.32 pg/mg protein (Range: 461.76 to 4,805.85 pg/mg protein). Average PD-L1 concentration from non NPC specimens was 612.43±314.04 pg/mg protein (Range: 326.87 to 1,155.19 pg/mg protein). ConclusionsFrom this preliminary result, PD-L1 protein (both in tumor cells and in immune cells) from NPC specimens has been shown to be elevated compared to normal nasopharyngeal epithelium. This finding indicate that further work is warranted to test anti PD-L1 drug for NPC patients that has failed all the standard treatment. Furthermore, our upcoming work would be exploring relationship between PD-L1, EBV, and tumor microenvironment from our NPC specimens. Legal entity responsible for the studyThe authors. FundingUniversity of Indonesia. DisclosureAll authors have declared no conflicts of interest.

Ultrasensitive Electrochemiluminescence Aptasensor for Assessment of Protein Heterogeneity in Small Cell Population.

Development of sensitive method for assay of proteins secreted from small cell population is important for the assessment of cellular heterogeneity. Here, we developed an ultrasensitive electrochemiluminescence (ECL) aptasensor for secreted protein in small cell population of RAW 264.7 cells for cellular heterogeneity assessment. Tumor necrosis factor-alpha (TNF-α) was chosen as target secreted protein, and specific anti-TNF-α aptamer was chosen as molecule recognition element, while dichlorotris (1,10-phenanthroline) ruthenium(II) hydrate (Ru(phen)32+) was chosen as ECL signal compound. An ECL signal probe was prepared by combining aptamer, Ru(phen)32+, and graphene oxide (GO) through electrostatic interaction and π-π stacking interaction. An aptasensor was fabricated by self-assembly specific aptamer on gold nanoparticles modified electrode for the detection of TNF-α using sandwich assay. High sensitivity and good selectivity were obtained for TNF-α. Parallel analysis was conducted to detect TNF-α secreted by RAW 264.7 cells stimulated by either lipopolysaccharides (LPS) or lupeol for assessing the cell heterogeneity in small cell population and for estimating the intracellular signal transduction in inflammation. Compared to ELISA, our ECL method offers both high sensitivity and good accuracy. The distinct heterogeneity among the secreted protein in small cell population, for the first time, was observed and further correlated to the cell's nature of cluster growth. The developed ECL method incorporating the amplification of nanomaterial and selectivity of aptamer is promising for sensitive and selective analysis of biomolecules in small cell population.

Michaelis-Menten Parameters Characterization of Commercial Papain Enzyme “Paya”

The use of enzymes is increasing nowadays, both for research and for the industry. This happens due to the advances in various fields such as fermentation technology, genetic engineering, and enzyme applications technology. One kind of enzymes that is very widely used and plays an important role in biotechnology and industrial applications is a protease enzyme, especially papain enzyme. Papain is a proteolytic enzyme which is isolated from papaya fruit sap tapping, or it could be derived from the leaves of papaya (Carica papaya L.). Actually, in the market, there is already a commercial papain enzyme called "Paya" which serves as a meat tenderizer with a relatively low price, but the enzyme papain was not pure and has unknown activity. Therefore, this study aims to examine the concentration of dissolved papain enzyme and casein as the substrate. By using the Lowry protein assay method, the initial concentration of papain enzyme and casein respectively 20,000 ppm. The result of protein assay we found that the concentration of dissolved papain enzyme and dissolved casein are respectively 71.069 ppm and 764.8276 ppm. The results from the first experiment are used for the second experiment which is to measure and determine the enzyme kinetics parameters Km and Vmax of that commercial papain enzyme with casein as the substrate. From this research, we found that the Km and Vmax of this commercial papain enzyme respectively 248.68 ppm dan 1.514 ppm casein/minute.

An historical perspective of the discovery of titin filaments.

This review takes readers back to 1949, when two Australian scientists, Draper and Hodge, reported the first high-resolution electron microscopy images of striated muscle. In 1953, Jean Hanson and Hugh Huxley published phase-contrast microscopy and electron microscopy images that established the filamentous nature of the sarcomere, namely the myosin-containing thick filaments and actin-containing thin filaments. They discussed a putative third filament system, possibly a thinner actin-containing S filament, that appeared to connect one Z disc to the next. The next year, two back-to-back papers appeared in Nature, the first by Andrew Huxley and Rolf. Niedergerke, the second by Hugh Huxley with Jean Hanson. Independently, they proposed the sliding of actin filaments and myosin filaments. These two filaments quickly became firmly established in the literature and, even today, they remain the basis for the sliding filament hypothesis. The putative third filament concept was dropped, mainly through the lack of evidence but also because it was difficult to accommodate in the hypothesis where two sets of filaments maintained their lengths constant while sliding produced sarcomere shortening. The view that actin and myosin comprise more than 80% of the myofibril proteins also made it difficult to accommodate a major new protein. In the following years, using selective extraction of myosin and actin, dos Remedios (PhD thesis, University of Sydney, 1965) revealed a residual filament system in the sarcomere, and, once again, a third filament system re-entered the literature. Filaments were reported crossing the gap between the ends of thick and thin filaments in highly stretched muscle fibres. These and other early studies necessarily focussed on light and electron microscopy, and set the scene for investigations into the chemical nature and biophysical functions of the third filament system for striated muscles. Further progress had to wait for the improvement and/or development of a number of techniques. For example, in 1970, Laemmli (Nature 227:680-685, 1970) published an often cited method for improving SDS polyacrylamide gel electrophoresis. The Lowry et al. (J Biol Chem 193:265-275, 1951) protein assay method developed in 1950 was both unstable and insensitive in comparison, but we had to wait until 1976 for the development of the Bradford method (1976). Atomic force microscopy was not known before 1986, but it eventually enabled the direct measurement of single molecules of titin. This extraordinarily large (>106Da) elastic protein became known as connectin (Maruyama in J Biochem 80:405-407, 1976) and was subsequently named titin (Wang et al. in Proc Natl Acad Sci U S A 76:3698-3702, 1979). Prior to the discovery of titin/connectin, biophysicists found it difficult to understand howa single polypeptide chain could could stretch from the Z disc to the M line, a distance of more than 1μm. It was quite literally the 'elephant in the room'. In this review, we follow the trail of microscopy-based reports that led to the emergence of what is now known and accepted as titin, an elastic third filamentous protein that is the focus of this Special Issue.

Sensitive double antibody sandwich ELISA for the quantification of phosvitin

ABSTRACTAn effective double antibody sandwich ELISA (DAS-ELISA) method based on monoclonal (mAb) and chicken egg yolk IgY antibodies was developed to determine phosvitin (PV) content in therapeutic and functional products. Leghorn laying hens were immunized with purified PV to produce anti-PV IgY antibody in the egg yolk. High anti-PV IgY titer obtained from the egg yolks collected during 4–10 weeks of the immunization period contained approximately 6.2% of specific anti-PV IgY in total IgY. The PV detection range of the DAS-ELISA and biotinylated DAS-ELISA was 16.8–90 and 7.5–40 ng/mL, respectively. However, biotinylated DAS-ELISA was the better method for PV quantification in terms of accuracy and sensitivity. This highly efficient PV detection method may recuperate the performance of the existing protein assay methods as well as facilitate future research on PV bioactivities and applications.

Colorimetric detection of proteins based on target-induced activation of aptazyme

The detection of protein is vital to fundamental research as well as practical applications. However, most detection methods depend on antibody-based assays which are faced with many shortcomings. Herein, we propose a colorimetric method for protein assays based on target-triggered activation of aptazyme, which may offer simple, rapid and cost-effective detection of the target protein. In this method, the conformation change of aptazyme induced by target protein is designed to be associated with aptazyme activation. Consequently, in the presence of the target protein, the designed DNA linkers will be cleaved into two fragments that fail to cross-link gold nanoparticles (GNPs), thus the color of GNP solution remains red, while the color will be changed in the absence of the target. Because of the advantages of aptazyme such as economic synthesis, stable, easy modification and its ability to accomplish signal recognition and signal amplification simultaneously, the method is thermostable, simple and cost-efficient. In this work, we have taken the detection of vascular endothelial growth factor (VEGF) as an example, which can present an analytical performance with as low as 0.1 nM detection limit, spanning a detection range of 3 orders of magnitude. What is more, the principle of this proposed new method can be extended as a universal assay method not only for the detection of analytes which have an aptamer but also for those analytes that have ligands.

A single step protein assay that is both detergent and reducer compatible: The cydex blue assay.

Determination of protein concentration is often an absolute prerequisite in preparing samples for biochemical and proteomic analyses. However, current protein assay methods are not compatible with both reducers and detergents, which are however present simultaneously in most denaturing extraction buffers used in proteomics and electrophoresis, and in particular in SDS electrophoresis. It was found that inclusion of cyclodextrins in a Coomassie blue-based assay made it compatible with detergents, as cyclodextrins complex detergents in a 1:1 molecular ratio. As this type of assay is intrinsically resistant to reducers, a single-step assay that is both detergent and reducer compatible was developed. Depending on the type and concentration of detergents present in the sample buffer, either beta-cyclodextrin or alpha-cyclodextrin can be used, the former being able to complex a wider range of detergents and the latter being able to complex higher amounts of detergents due to its greater solubility in water. Cyclodextrins are used at final concentrations of 2-10mg/mL in the assay mix. This typically allows to measure samples containing as little as 0.1mg/mL protein, in the presence of up to 2% detergent and reducers such as 5% mercaptoethanol or 50mM DTT in a single step with a simple spectrophotometric assay.