Journal: Molecular Biology

Article Title: Adaptation of Microarray Assay for Serum Amyloid a Analysis in Human Serum

doi: 10.1134/S0026893322020145

Figure Lengend Snippet: Fluorescent signals from pads with immobilized antibodies to SAA (SAA1 and SAA15) and from empty gel after analysis of SAA solution (10 μg/mL).

Article Snippet: We used the following reagents: Tween-20, polyvinyl alcohol (PVA) 50 kDa, polyvinylpyrrolidone (PVP) 360 kDa, streptavidin-Cy5 conjugate (Streptavidin-Cy5) (GE Healthcare, USA), phosphate buffered saline pH 7.4 (PBS), PBS with 0.01% Tween-20 (PBST); dilution buffer (PBS, 0.14% PVA, 0.14% PVP), rabbit blood serum (Biosera, France), goat blood serum (Biosera), mouse blood serum (Biosera), monoclonal antibodies to SAA (clone SAA1, clone SAA15) and their conjugates with biotin (HyTest, Russia), and human SAA (HyTest).

Techniques:

Journal: Molecular Biology

Article Title: Adaptation of Microarray Assay for Serum Amyloid a Analysis in Human Serum

doi: 10.1134/S0026893322020145

Figure Lengend Snippet: Dependence of the fluorescent signal on the concentration of SAA in the analysis of antigen dissolved in a mixture of animal sera (mouse, goat, rabbit). Fluorescence values from pads containing anti-SAA antibodies are shown. The signal from the empty gel in all experiments did not exceed 0.25 arb. units.

Article Snippet: We used the following reagents: Tween-20, polyvinyl alcohol (PVA) 50 kDa, polyvinylpyrrolidone (PVP) 360 kDa, streptavidin-Cy5 conjugate (Streptavidin-Cy5) (GE Healthcare, USA), phosphate buffered saline pH 7.4 (PBS), PBS with 0.01% Tween-20 (PBST); dilution buffer (PBS, 0.14% PVA, 0.14% PVP), rabbit blood serum (Biosera, France), goat blood serum (Biosera), mouse blood serum (Biosera), monoclonal antibodies to SAA (clone SAA1, clone SAA15) and their conjugates with biotin (HyTest, Russia), and human SAA (HyTest).

Techniques: Concentration Assay, Fluorescence

Journal: Molecular Biology

Article Title: Adaptation of Microarray Assay for Serum Amyloid a Analysis in Human Serum

doi: 10.1134/S0026893322020145

Figure Lengend Snippet: Fluorescent image of pads with immobilized antibodies to SAA after analysis of analytes containing SAA. (a) Analysis of serum containing SAA, short-term incubation with the sample. (b) Analysis of serum containing SAA, incubation with the sample for 20 h. (c) Analysis of SAA solution in dilution buffer (PBS, 0.14% PVA, 0.14% PVP), incubation with the sample for 20 h.

Article Snippet: We used the following reagents: Tween-20, polyvinyl alcohol (PVA) 50 kDa, polyvinylpyrrolidone (PVP) 360 kDa, streptavidin-Cy5 conjugate (Streptavidin-Cy5) (GE Healthcare, USA), phosphate buffered saline pH 7.4 (PBS), PBS with 0.01% Tween-20 (PBST); dilution buffer (PBS, 0.14% PVA, 0.14% PVP), rabbit blood serum (Biosera, France), goat blood serum (Biosera), mouse blood serum (Biosera), monoclonal antibodies to SAA (clone SAA1, clone SAA15) and their conjugates with biotin (HyTest, Russia), and human SAA (HyTest).

Techniques: Incubation

Journal: eLife

Article Title: Serum amyloid A is a retinol binding protein that transports retinol during bacterial infection

doi: 10.7554/eLife.03206

Figure Lengend Snippet: ( A ) Retinol exhibits intrinsic fluorescence that is enhanced upon binding to proteins through energy transfer from tryptophan residues. All- trans -retinol was titrated into mSAA3 and fluorescence emission was monitored following excitation at 348 nm. The chemical structure of retinol is shown. ( B ) All- trans -retinol was titrated into hSAA1, mSAA1, mSAA3, human transferrin (hTfr; negative control), and apolipoprotein A1 (ApoA1; negative control). Binding was quantified by monitoring retinol fluorescence at 460 nm following excitation at 348 nm as in ( A ). Plots are representative of five independent experiments. ( C ) Retinoic acid lacks intrinsic fluorescence, but can quench intrinsic protein fluorescence due to energy transfer from tryptophan residues . All- trans -retinoic acid was titrated into mSAA3 and fluorescence quenching was monitored following excitation at 296 nm. The chemical structure of retinoic acid is shown. ( D ) All- trans -retinoic acid was titrated into hSAA1, mSAA1, mSAA3, hTfr, and ApoA1. Fluorescence emission was monitored at 334 nm with excitation at 296 nm as in ( C ). Plots are representative of three independent experiments. ( E ) K d s were calculated from the binding assay data plotted in ( B ) and ( D ) and were derived from three independent experiments. nd, not determined. Additional ligand binding measurements are provided in . DOI: http://dx.doi.org/10.7554/eLife.03206.007

Article Snippet: Zinc-fixed, paraffin embedded tissue sections were stained with anti-SAA antiserum raised against purified recombinant mSAA1 and detected using a goat anti-rabbit IgG Cy3 conjugate (Biomeda).

Techniques: Fluorescence, Binding Assay, Negative Control, Derivative Assay, Ligand Binding Assay

Journal: eLife

Article Title: Serum amyloid A is a retinol binding protein that transports retinol during bacterial infection

doi: 10.7554/eLife.03206

Figure Lengend Snippet: ( A – C ) Retinyl acetate ( A ), β-carotene ( B ), and retinyl palmitate ( C ) were titrated into hSAA1, mSAA1, mSAA3, hTfr (negative control) and ApoA1 (negative control), and fluorescence quenching was monitored at 334 nm with excitation at 296 nm. Plots are representative of three independent experiments. The K d s are averages of the values derived from the three experiments. ( D ) Competitive inhibition of retinol binding by cholesterol was quantified. Saturating concentrations of retinol were added to hSAA1, mSAA1, and mSAA3, and fluorescence quenching was monitored as in ( A – C ). 10 μM cholesterol was added into the assay and inhibition of fluorescence quenching by retinol was monitored. Values are the average ± SEM of triplicate experiments. DOI: http://dx.doi.org/10.7554/eLife.03206.009

Article Snippet: Zinc-fixed, paraffin embedded tissue sections were stained with anti-SAA antiserum raised against purified recombinant mSAA1 and detected using a goat anti-rabbit IgG Cy3 conjugate (Biomeda).

Techniques: Negative Control, Fluorescence, Derivative Assay, Inhibition, Binding Assay