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

Abstract

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>

Modified Desolvation Method Enables Simple One-Step Synthesis of Gelatin Nanoparticles from Different Gelatin Types with Any Bloom Values

“…As we mentioned in Section 3.3 , our goal was to prepare relatively small nanoparticles, less than 200 nm, which is possible by using smaller gelatin concentrations for all tested gelatin types. Data obtained in the course of optimization experiments and our previous results [ 30 ] both demonstrate that gelatin nanoparticles can be prepared at high starting gelatin concentrations. Variation of pH and volume of the desolvating agent is a possible way to decrease the size of nanoparticles when the concentration of gelatin is high.…”

“…At the same time, homogeneous suspensions of gelatin nanoparticles with polydispersity indices lower than 0.1 were formed when mixing was performed on the rotator ( Figure 1 ). We did not perform experiments with higher gelatin concentrations, but recently we successfully prepared gelatin nanoparticles using 30 mg/mL gelatin solution which was desolvated under short gentle mixing [ 30 ].…”

“…Protein nanoparticles cross-linked with glutaraldehyde emit fluorescence when excited by UV or visible light [ 30 , 61 ]. Autofluorescence of gelatin nanoparticles can be explained by the presence of C=C (resulting from glutaraldehyde polymerization) and C=N bonds (in the Shiff bases) in nanoparticles’ structure [ 62 ].…”

“…Intensive stirring promotes gelatin aggregation. Using a simple one-step stirring-free approach we previously prepared stable and homogeneous gelatin nanoparticles from gelatin with bloom values as low as 75 [ 30 ]. Based on these findings we intended to develop a facile method for nanoparticle preparation from any type of gelatin with any bloom number.…”

One-Pot Biosynthesis of l-Aspartate from Maleic Anhydride via a Thermostable Dual-Enzyme System under High Temperature

“…Buffer A (50 mM NaH 2 PO 4 , 30 mM NaCl, 10 mM imidazole, pH 7.5) was used to wash away nontarget proteins, and then the target proteins were eluted using buffer B (50 mM Na 2 HPO 4 , 300 mM NaCl, 150 mM imidazole, pH 7.5). The protein concentration was measured by the Bradford method, 30 and the protein molecular mass was evaluated by SDS-PAGE.…”