Biotinylated cages were incubated in TBS (Tris-HCl 50 mM, NaCl 150 mM, pH 7.8) or in culture medium supplemented with 10% FBS at 37 °C for different times. Each sample was then digested with proteinase K (100 μg/mL) for 1 h at 37 °C, and analyzed by DNA blot, as previously described [21 (link)]. Biotin detection was carried out using streptavidin–HRP (horseradish peroxidase) (Abcam Inc., Toronto, ON, Canada), and visualized by enhanced chemiluminescence (ECL Extend, Euroclone, Devon, UK). For image processing and densitometric analysis, photographic films were digitized by scanning and bands analyzed by ImageJ software.
Streptavidin hrp horseradish peroxidase
Manufactured by Abcam
Sourced in Canada
Streptavidin-HRP (Horseradish Peroxidase) is a conjugate of streptavidin and horseradish peroxidase. Streptavidin has a high affinity for biotin, and the HRP enzyme catalyzes a colorimetric reaction. This product can be used in various bioanalytical techniques that utilize the biotin-streptavidin interaction.
Automatically generated - may contain errors
6 protocols using streptavidin hrp horseradish peroxidase
1
Biotinylated Cage Analysis by DNA Blot
2
Intracellular Stability of Biotinylated Nanocages
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For in vitro stability, biotinylated nanocages were incubated in culture medium supplemented with 10% FBS at 37 °C for different times. Each sample was then digested with proteinase K (100 µg/mL) for 1 h at 37 °C and analyzed by DNA blot, as described [8 (link)].
For evaluating the intracellular stability, cells were plated in 48-well plates at a density of 3 × 104 cells/well and grown for 24 h before treatments. After incubation with DNA nanocages for different time periods, cells were lysed, centrifuged, digested with proteinase K, and analyzed with DNA blot [8 (link)]. Biotinylated nanocages detection was carried out using streptavidin–HRP (horseradish peroxidase) (Abcam Inc., Toronto, ON, Canada), and visualized using enhanced chemiluminescence (ECL Extend, Euroclone, Devon, UK). For image processing, photographic films were digitized and densitometric analysis was performed with ImageJ 1.52a software.
For evaluating the intracellular stability, cells were plated in 48-well plates at a density of 3 × 104 cells/well and grown for 24 h before treatments. After incubation with DNA nanocages for different time periods, cells were lysed, centrifuged, digested with proteinase K, and analyzed with DNA blot [8 (link)]. Biotinylated nanocages detection was carried out using streptavidin–HRP (horseradish peroxidase) (Abcam Inc., Toronto, ON, Canada), and visualized using enhanced chemiluminescence (ECL Extend, Euroclone, Devon, UK). For image processing, photographic films were digitized and densitometric analysis was performed with ImageJ 1.52a software.
3
Quantifying Cellular Uptake of H4-NCs
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Cells were plated in 48-well plates at a density of 3 × 104 cells/well and grown 24 h before the experiment. Cells, incubated with H4-NCs at different concentrations and times (as indicated in each experiment) were then lysed, centrifuged, digested with proteinase K, and analyzed by DNA blot, as previously described [22 (link)]. Biotin detection was carried out, using streptavidin-HRP (Horseradish Peroxidase) (Abcam Inc, Toronto, ON, Canada), and visualized by enhanced chemiluminescence (ECL Extend, Euroclone, Devon UK). For purification of H4-NCs from conditioned medium, medium was collected from each well, cleared from cellular debris by centrifugation at 10,000 rpm for 15 min, digested with proteinase K, and analyzed by DNA blot [22 (link)]. Input samples of H4-NCs are DNA structures added to cell-culture medium and immediately digested with proteinase K and processed for DNA blot.
4
SARS-CoV-2 RBD Binding Assay
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25ng of hACE2-Fc fusion protein expressed in HEK293 cells were adhered to high-capacity binding, 96 well-plates (Corning) overnight in PBS. Plates were blocked with 5% BSA in PBS containing Tween-20 (PBS-T) for 1 h at room temperature (RT). Blocking solution was discarded and 5-fold dilutions of 6xHis-tagged RBDs in PBS were added to wells and incubated for 1 h at RT. Plates were then washed three times with PBS-T. Anti-polyhistidine IgG-Biotin (Abcam) in PBS-T was added to each and incubated for 1 h at RT. Plates were then washed three times with PBS-T. Streptavidin-HRP (horseradish peroxidase) (Abcam) in PBS-T was added to each and incubated for 1 h at RT. Plates were then washed three times with PBS-T Plates were developed using 1-Step Ultra TMB (3,3′,5,5′-tetramethylbenzidine) substrate (ThermoFisher), stopped with sulfuric acid and immediately read using a plate reader at 450nm. Data were plotted using Prism 9 (GraphPad Software) and affinities determined by applying a nonlinear regression model.
5
Detecting Biotinylated Nanocarriers in Cells
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Cells were plated in 48-well plates at a density of 3 × 104 cells/well and grown in folate-free RPMI 1640 (Sigma Aldrich, St Louis, MO, USA) supplemented with 10% FBS for 24 h. Experiments were performed in folate-free RPMI 1640 medium with 2% FBS. Cells were lysed, centrifuged, digested with proteinase K, and analyzed by DNA blot, as described earlier25 (link). Detection of biotinylated-NCs was carried out using streptavidin-HRP (Horseradish Peroxidase) (Abcam Inc., Toronto, ON, Canada, Ab7403) and visualized by enhanced chemiluminescence (ECL Extend, Euroclone, Devon, UK). Input samples of NCs are DNA structures added to cell culture medium and immediately digested with proteinase K and processed for DNA blot.
6
Quantifying DNA Nanocage Uptake
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Cells were plated in 48 well plates at a density of 5x10 4 cells/well and grown 24 hours in folate-free RPMI 1640 supplemented with 10% FBS. Cells were incubated with Bio-Fol DNA cages at different concentration and time (as indicated in each experiment). After incubation, cells were lysed, centrifuged, digested with proteinase K and analysed by DNA blot, as previously described. 10 Biotin detection was carried out using streptavidin-HRP (Horseradish Peroxidase) (Abcam) and visualized by enhanced chemiluminescence (ECL Plus, Euroclone). For image processing and densitometric analyses, photographic films were digitized by scanning. Bands were analysed with ImageJ software.
DNA nanocages were purified from conditioned medium after incubation with cells for different times at 37 °C. Conditioned medium was collected from each well, cleared from cellular debris by centrifugation at 10.000 rpm for 15 min and analysed by DNA blot. 10 For the preparation of DNA nanocage input samples, cages added to cell culture medium were immediately digested with proteinase K (100 µg/ml) for 1 h at 37 °C and protein digestion was stopped by adding phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 5 mM.
DNA nanocages were purified from conditioned medium after incubation with cells for different times at 37 °C. Conditioned medium was collected from each well, cleared from cellular debris by centrifugation at 10.000 rpm for 15 min and analysed by DNA blot. 10 For the preparation of DNA nanocage input samples, cages added to cell culture medium were immediately digested with proteinase K (100 µg/ml) for 1 h at 37 °C and protein digestion was stopped by adding phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 5 mM.
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