scholarly journals A simple method for non-denaturing purification of biotin-tagged proteins through competitive elution with free biotin

BioTechniques ◽  
2020 ◽  
Vol 68 (1) ◽  
pp. 41-44
Author(s):  
Kui Lin ◽  
Qin Yan ◽  
Audrey Mitchell ◽  
Natasha Funk ◽  
Catherlin Lu ◽  
...  
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Protein Purification ◽  
Recovery Rate ◽  
Bovine Serum ◽  
Protein Denaturation ◽  
Simple Method ◽  
Agarose Beads ◽  
Elution Buffer ◽  
Neutral Conditions

The use of avidin or streptavidin in the purification of biotinylated proteins has been highly restricted due to the harsh and denaturing elution conditions. Here, we use biotinylated bovine serum albumin as a working model to demonstrate a simple and rapid method for biotin-tagged protein purification under non-denaturing conditions. The biotinylated bovine serum albumin is specifically bound to the anti-biotin antibody agarose beads and competitively eluted with free biotin under near-neutral conditions. The optimized elution conditions include using 4 mg/ml biotin (pH 8.5) as the elution buffer and allowing the buffer to incubate with agarose beads for 30 min prior to elution. The elution recovery rate is over 85% without apparent protein denaturation. The method is applicable for both immunoprecipitation and column chromatography.

scholarly journals Biogenic Ferrihydrite Nanoparticles Produced by Klebsiella oxytoca: Characterization, Physicochemical Properties and Bovine Serum Albumin Interactions

Nanomaterials ◽  
2022 ◽  
Vol 12 (2) ◽  
pp. 249
Author(s):  
Nicoleta Cazacu ◽  
Claudia G. Chilom ◽  
Sorina Iftimie ◽  
Maria Bălășoiu ◽  
Valentina P. Ladygina ◽  
...  
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Protein Denaturation ◽  
Klebsiella Oxytoca ◽  
Resonance Energy ◽  
Elemental Content ◽  
X Ray ◽  
Synthesis Of Nanoparticles ◽  
Vis Spectroscopy

The synthesis of nanoparticles inside microorganisms is an economical alternative to chemical and physical methods of nanoparticle synthesis. In this study, ferrihydrite nanoparticles synthesized by Klebsiella oxytoca bacterium in special conditions were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDS), small-angle X-ray (SAXS), UV-Vis spectroscopy, fluorescence, fluorescence resonance energy transfer (FRET), and molecular docking. The morphology and the structure of the particles were characterized by means of SEM and SAXS. The elemental content was determined by means of the EDS method. The absorption properties of the ferrihydrite nanoparticles were investigated by UV-Vis spectroscopy. The binding mechanism of the biogenic ferrihydrite nanoparticles to Bovine Serum Albumin (BSA) protein, studied by fluorescence, showed a static and weak process, combined with FRET. Protein denaturation by temperature and urea in the presence of the ferrihydrite nanoparticles demonstrated their influence on the unfolding process. The AutoDock Vina and UCSF Chimera programs were used to predict the optimal binding site of the ferrihydrite to BSA and to find the location of the hydrophobic cavities in the sub-domain IIA of the BSA structure.

Synthesis, Anti-Arthritic, and Anti-Inflammatory Activity of N-Tosyl aza Cyclophanes

2012 ◽  
Vol 65 (2) ◽  
pp. 186 ◽  
Author(s):  
Perumal Rajakumar ◽  
Ramar Padmanabhan
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Blood Cells ◽  
Bovine Serum ◽  
Protein Denaturation ◽  
Inflammatory Activity ◽  
Stabilization Method ◽  
Membrane Stabilization ◽  
Human Red Blood Cells ◽  
Anti Inflammatory

The synthesis of novel N-tosyl tetraaza cyclophanes and N-tosyl diaza cyclophane incorporating m-terphenyl as spacer units is described. Anti-arthritic activity was studied by inhibition of the protein denaturation method (bovine serum albumin). All the N-tosyl aza cyclophanes exhibit excellent anti-arthritic activity. Anti-inflammatory activity of the synthesized cyclophanes was investigated using the human red blood cells (HRBC) membrane stabilization method and some of the N-tosyl aza cyclophanes exhibited good anti-inflammatory activity.

scholarly journals Adsorption of bovine serum albumin on silver surfaces enhances the release of silver at pH neutral conditions

2015 ◽  
Vol 17 (28) ◽  
pp. 18524-18534 ◽  
Author(s):  
X. Wang ◽  
G. Herting ◽  
I. Odnevall Wallinder ◽  
E. Blomberg
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Surface Complexation ◽  
Surface Complexes ◽  
Silver Substrate ◽  
Neutral Conditions ◽  
Silver Surfaces

Enhanced release of silver in the presence of BSA due to surface complexation between BSA and the silver substrate, followed by exchange of surface complexes with BSA molecules in solution.

scholarly journals Label-Free Impedimetric Immunosensors Modulated by Protein A/Bovine Serum Albumin Layer for Ultrasensitive Detection of Salbutamol

Sensors ◽  
2020 ◽  
Vol 20 (3) ◽  
pp. 771
Author(s):  
Chia-Hung Lin ◽  
Ming-Jie Lin ◽  
Jie-De Huang ◽  
Yu-Sheng Chuang ◽  
Yu-Fen Kuo ◽  
...  
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Electrochemical Impedance ◽  
Protein A ◽  
Great Promise ◽  
Igg Antibody ◽  
Simple Method ◽  
Sensing Properties ◽  
Relative Standard

The sensing properties of immunosensors are determined not only by the amount of immobilized antibodies but also by the number of effective antigen-binding sites of the immobilized antibody. Protein A (PA) exhibits a high degree of affinity with the Fc part of IgG antibody to feasibly produce oriented antibody immobilization. This work proposes a simple method to control the PA surface density on gold nanostructure (AuNS)-deposited screen-printed carbon electrodes (SPCEs) by mixing concentration-varied PA and bovine serum albumin (BSA), and to explore the effect of PA density on the affinity attachment of anti-salbutamol (SAL) antibodies by electrochemical impedance spectroscopy. A concentration of 100 μg/mL PA and 100 μg/mL BSA can obtain a saturated coverage on the 3-mercaptoproponic acid (MPA)/AuNS/SPCEs and exhibit a 50% PA density to adsorb the amount of anti-SAL, more than other concentration-varied PA/BSA-modified electrodes. Compared with the randomly immobilized anti-SAL/MPA/AuNS/SPCEs and the anti-SAL/PA(100 μg/mL):BSA(0 μg/mL)/MPA/AuNS/SPCE, the anti-SAL/PA(100 μg/mL): BSA(100 μg/mL)/MPA/AuNS/SPCE-based immunosensors have better sensing properties for SAL detection, with an extremely low detection limit of 0.2 fg/mL and high reproducibility (<2.5% relative standard deviation). The mixture of PA(100 μg/mL):BSA(100 μg/mL) for the modification of AuNS/SPCEs has great promise for forming an optimal protein layer for the oriented adsorption of IgG antibodies to construct ultrasensitive SAL immunosensors.

scholarly journals A DSC study of zinc binding to bovine serum albumin (BSA)

2007 ◽  
Vol 72 (4) ◽  
pp. 331-337 ◽  
Author(s):  
Sanja Ostojic ◽  
Vida Dragutinovic ◽  
Miodrag Kicanovic ◽  
Branislav Simonovic
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Thermal Denaturation ◽  
Protein Unfolding ◽  
Protein Denaturation ◽  
Binding Constants ◽  
Zinc Binding ◽  
Scanning Calorimetry ◽  
The Stability

The thermal denaturation of bovine serum albumin (BSA) is a kinetically and thermodynamically controlled process. The effects of zinc binding to bovine serum albumin (BSA), followed by differential scanning calorimetry (DSC), were investigated in this work, with the purpose of obtaining a better understanding of the albumin/zinc interaction. From the DSC curves, the thermodynamic parameters of protein denaturation were obtained, i.e., the temperature of thermal transition maximum (T m), calorimetric enthalpy (?Hcal), van't Hoff enthalpy (?HvH), the number of binding sites (I, II), the binding constants for each binding site (K bI, K bII) and the average number of ligands bound per mole of native protein X N. The thermodynamic data of protein unfolding showed that zinc binding to bovine serum albumin increases the stability of the protein (higher values of ?Hcal) and the different ratio ?Hcal/?HvH indicates the perturbation of the protein during thermal denaturation.

scholarly journals Interference of freeze-dried Hepatitis A vaccine (H2 strain) on testing bovine serum albumin content by ELISA

2013 ◽  
Vol 2 (2) ◽  
pp. 21
Author(s):  
Yueqing Chen ◽  
Jun Chen ◽  
Zhuo Chen ◽  
Feng Zhang ◽  
Hualiang Xia ◽  
...  
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Glutamic Acid ◽  
Recovery Rate ◽  
Bovine Serum ◽  
Hepatitis A ◽  
Interference Effects ◽  
Elisa Kit ◽  
Freeze Dried ◽  
Albumin Content

<p><strong>Objective</strong> To carry on preliminary research on interference effects of freeze-dried attenuated Hepatitis A vaccine（H2 strain）on testing residual Bovine Serum Albumin content by ELISA kit. <strong>Methods</strong> Interference test was conducted according to Appendix VIII, Chinese Pharmacopoeia (Vol III,2010 edi), and the interference level and its possible resources were explored.<strong> Results</strong> The protective additives of HAV live vaccine do have certain interference on determining residual BSA content by ELISA kit, among which Cysteine and Glutamic acid might play the leading roles. The general BSA recovery rate is 54% to 85%. We assessed the interference degree by using series of different concentration of single amino acid ingredient: cysteine (0 to 0.2%),glutamic acid(0 to 0.8%). We found BSA recovery rate always higher than 70% with 0 to 2mg/ml (0.2%) cysteine, lower with 0.5 to 2mg/ml than with 0 to 0.5mg/ml and did not differ between 0.5 and 2mg/ml cysteine; And the BSA recovery rates were up to 100% if we applied Glutamic acid (concentration not higher than 0.1%) as an interference. It deserves to be mentioned that the interference effects of Glutamic acid become more stronger along with concentration rising from 0.1% to the peak 0.8%(8mg/ml),the test results showed to be not higher than 2.5ng/ml when we set 20ng/ml BSA standards as a target sample, so that the recovery rate is not more than 12.5%. <strong>Conclusion</strong> The real value of residual BSA content of HAV vaccine should be reported based on the extent of interference. Here when the result appeared to be negative by ELISA we would report as &lt;2.5 ng/ml and as &lt; 5ng/ml if the testing value showed between 0 and 3ng/ml. And what we have done might be a reference to the other enterprises on optimizing the protocol for cryopreservation or evaluating the residual BSA content results.</p>

Improvement of an artificial test substrate technique for evaluation of em immunocytochemical labeling

1987 ◽  
Vol 45 ◽  
pp. 762-763
Author(s):  
G. D. Gagne ◽  
M. F. Miller
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Artificial Substrate ◽  
Chemical Fixation ◽  
As If

We recently described an artificial substrate system which could be used to optimize labeling parameters in EM immunocytochemistry (ICC). The system utilizes blocks of glutaraldehyde polymerized bovine serum albumin (BSA) into which an antigen is incorporated by a soaking procedure. The resulting antigen impregnated blocks can then be fixed and embedded as if they are pieces of tissue and the effects of fixation, embedding and other parameters on the ability of incorporated antigen to be immunocyto-chemically labeled can then be assessed. In developing this system further, we discovered that the BSA substrate can also be dried and then sectioned for immunolabeling with or without prior chemical fixation and without exposing the antigen to embedding reagents. The effects of fixation and embedding protocols can thus be evaluated separately.

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