Determination of total protein content in biomedical products by the PDMS-assisted lab-in-a-syringe assay using 3D printed scaffolds removal

The aim of our work was to develop a low-cost, portable device for the fast and easy determination of total protein content by using PDMS-based lab-in-a-syringe technology with removal of 3D-printed channels. We proposed two designs with a one-step PDMS curing and a two-step PDMS-curing fabrication procedure. The one-step PDMS microdevices were found to be the best in the view of preparation, repeatability, and stability of the reagent. This design was then applied for the determination of total protein content in biomedical products using the Bradford assay.

An organic solvent-based protocol for depletion of abundant proteins in plasma of Leprosy patients

In present study protein was purified from human plasma with the method involving organic solvents. Organic solvents like chloroform, methanol and isopropanol remove the lipoproteins and glycoproteins whereas TCA and Acetone are known to deplete the abundant proteins like albumins and globulins from the plasma of the blood. Purified protein was subjected to the estimation by Bradford Assay (96 well plate method). In this process a dye called Brilliant Blue forms complex with the purified protein of plasma because of which the maximum absorption of dye shifts to 595nm. So, the absorbance was observed at wavelength of 595nm. Purified protein along with unpurfled plasma was imposed on SDS-PAGE. SDS (sodium dodecyl sulphate) is an anionic detergent which first denatures the protein and then provides all the protein fragments a negative charge. After which polyacrylamide gel the separates the protein fragments on the basis of their molecular mass. Results of SDS-PAGE concluded that the protein was purified when its bands were compared to that of unpurfled plasma, which means the treatment with organic solvents is useful in purifying protein from plasma of leprosy patient.

Evaluation of 3 Common Methods for Effective Housefly Allergen Extraction

Background: Allergen extracts have been applied to treat allergic diseases. Accordingly, a housefly (Musca domestica) extract is commonly used to treat patients severely allergic to housefly. Objective: To evaluate 3 common methods, including grinding, sonication, and homogenization, for effective preparation of housefly allergen extracts. Methods: Housefly allergens were extracted from Musca domestica using 3 different methods, including grinding, sonication, and homogenization. Protein concentrations and profiles in the extracts were determined by Bradford assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Results: The protein concentrations of the extracts prepared by grinding (mean [SD], 911.3 [159.7] µg/µL) and sonication (mean [SD], 905.7 [188.6] µg/µL) as measured by Bradford assay were significantly higher than those prepared by homogenization (mean [SD], 674.5 [60.0] µg/µL). Moreover, SDS-PAGE showed more protein bands in the extracts prepared using grinding and sonication compared to those prepared using homogenization. Conclusions: In comparison to homogenization, both grinding and sonication methods are superior ways to prepare housefly allergen extracts as evidenced by the higher quantities and composition of proteins.

An Alternative Approach to Evaluate the Quality of Protein-Based Raw Materials for Dry Pet Food

The majority of dry pet food currently on the market is produced using fresh meats (FMs) and especially meat meals (MMs) as the main protein source. The transport and storage conditions of the raw materials, together with thermal and mechanical treatments in the case of MMs, may result in undesirable alterations of food products and their protein content. This study was conducted to analyze the protein component of three different kinds of raw materials used for dry pet food production, i.e., chicken, pork, and salmon. The quantitative analysis of the protein component was determined using the traditional Kjeldahl method and near-infrared (NIR) spectroscopy, and an alternative method, i.e., the Bradford assay, while the qualitative analysis was performed through SDS-PAGE, followed by Coomassie Blue staining. The amino acid (AA) profile was also evaluated by quadrupole time-of-flight liquid chromatography/mass spectrometry (Q-TOF LC/MS). In addition, the digestibility was tested through in vitro gastric and small intestine digestion simulation. Statistical analysis was performed by the Student’s t-test, and data are reported as mean ± SEM, n = 10 (p < 0.05). The results showed that the MMs are lower in quality compared to FMs, both in terms of protein bioavailability and digestibility, having a lower soluble protein (SP) content (chicken MM = 8.6 g SP/100 g dry sample; pork MM = 6.2 g SP/100 g dry sample; salmon MM = 7.9 g SP/100 g dry sample) compared to FMs (chicken FM = 14.6 g SP/100 g dry sample; pork FM = 15.1 g SP/100 g dry sample; salmon FM = 13.7 g SP/100 g dry sample). FMs appear, therefore, to be higher-quality ingredients for pet food production. Moreover, the Bradford assay proved to be a quick and simple method to better estimate protein bioavailability in the raw materials used for dry pet food production, thanks to its correlation with the in vitro digestibility.

Measuring the Concentration of Protein Nanoparticles Synthesized by Desolvation Method: Comparison of Bradford Assay, BCA Assay, hydrolysis/UV Spectroscopy and Gravimetric Analysis

Research paper on sunthesis of protein nanoparticles<div><br><div><b>Abstract</b></div><div>The desolvation technique is one of the most popular methods for preparing protein nanoparticles for medicine, biotechnology, and food applications. We fabricated 11 batches of BSA nanoparticles and 2 batches of gelatin nanoparticles by desolvation method. BSA nanoparticles from 2 batches were cross-linked by heating at +70 °C for 2 h; other nanoparticles were stabilized by glutaraldehyde. We compared several analytical approaches to measuring their concentration: gravimetric analysis, bicinchoninic acid assay, Bradford assay, and alkaline hydrolysis combined with UV spectroscopy. We revealed that the cross-linking degree and method of cross-linking affect both Bradford and BCA assay. Direct measurement of protein concentration in the suspension of purified nanoparticles by dye-binding assays can lead to significant (up to 50-60%) underestimation of nanoparticle concentration. Quantification of non-desolvated protein (indirect method) is affected by the presence of small nanoparticles in supernatants and can be inaccurate when the yield of desolvation is low. The reaction of cross-linker with protein changes UV absorbance of the latter. Therefore pure protein solution is an inappropriate calibrator when applying UV spectroscopy for the determination of nanoparticle concentration. Our recommendation is to determine the concentration of protein nanoparticles by at least two different methods, including gravimetric analysis.<div><br></div></div></div>

Measuring the Concentration of Protein Nanoparticles Synthesized by Desolvation Method: Comparison of Bradford Assay, BCA Assay, hydrolysis/UV Spectroscopy and Gravimetric Analysis

Research paper on sunthesis of protein nanoparticles<div><br><div><b>Abstract</b></div><div>The desolvation technique is one of the most popular methods for preparing protein nanoparticles for medicine, biotechnology, and food applications. We fabricated 11 batches of BSA nanoparticles and 2 batches of gelatin nanoparticles by desolvation method. BSA nanoparticles from 2 batches were cross-linked by heating at +70 °C for 2 h; other nanoparticles were stabilized by glutaraldehyde. We compared several analytical approaches to measuring their concentration: gravimetric analysis, bicinchoninic acid assay, Bradford assay, and alkaline hydrolysis combined with UV spectroscopy. We revealed that the cross-linking degree and method of cross-linking affect both Bradford and BCA assay. Direct measurement of protein concentration in the suspension of purified nanoparticles by dye-binding assays can lead to significant (up to 50-60%) underestimation of nanoparticle concentration. Quantification of non-desolvated protein (indirect method) is affected by the presence of small nanoparticles in supernatants and can be inaccurate when the yield of desolvation is low. The reaction of cross-linker with protein changes UV absorbance of the latter. Therefore pure protein solution is an inappropriate calibrator when applying UV spectroscopy for the determination of nanoparticle concentration. Our recommendation is to determine the concentration of protein nanoparticles by at least two different methods, including gravimetric analysis.<div><br></div></div></div>