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.
Ecl extend
Manufactured by Euroclone
Sourced in United Kingdom
ECL Extend is a chemiluminescent substrate used in western blotting applications to detect and visualize proteins. It provides a stable, long-lasting luminescent signal for sensitive protein detection.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin hrp horseradish peroxidase, by Abcam (4 mentions)
Hrp conjugated affinipure goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
β actin mouse mab, by Cell Signaling Technology (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Anti darpp32, by Merck Group (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
6 protocols using ecl extend
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
Western Blot Analysis of PTEN and Pdcd-4
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Cells were lysed and centrifuged as described previously31 (link). The supernatant fraction was analyzed by SDS-polyacrylamide gel electrophoresis in 10% acrylamide gels and blotted (Trans Blot Turbo Bio-Rad Laboratories). PTEN rabbit mAb (Cell Signaling Technologies, Danvers, MA, USA, cat. n. 138G6), Pdcd-4 mouse mAb (Santa Cruz Biotechnology, Dallas, Texas, USA, cat. n. SC-376430), and β-actin mouse mAb (Cell Signaling Technologies, Danvers, MA, USA, cat. n. 8H10D10) were used as primary antibodies. HRP-conjugated AffiniPure goat anti-mouse IgG (Jackson Immunoresearch, Cambridgeshire, UK, cat. n. 115-035-062) and HRP-conjugated AffiniPure donkey anti-rabbit IgG secondary antibody (Jackson Immunoresearch, Cambridgeshire, UK, cat. n. 711-035-152) were used as secondary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL Extend, Euroclone, Devon, UK). The ECL data were scanned and analyzed by ImageJ software. Band intensities were normalized to β-actin.
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
Quantitative Western Blot Analysis
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Western blot analysis was performed as previously described (Sarchielli et al., 2014) . Briefly, HSP cells were grown in F12 Coon's, treated with conditioned media and lysed in standard lysis buffer supplemented with protease inhibitor cocktail (Sigma Aldrich, Saint Louis, MO, USA). Aliquots containing 20 μg of proteins were loaded on SDS-PAGE, then transferred on poly-vinylidene difluoride membranes, blocked in 3% BSA and incubated with the following primary antibodies: anti-DARPP32 (1:1000, Sigma Aldrich) and anti-β actin (1:10000, Santa Cruz). The incubation with the primary antibodies was followed by peroxidase conjugated secondary IgG treatment and the reacted proteins were revealed by the enhanced chemiluminescence system (ECL extend, Euroclone, Milan, Italy). Image acquisition and densitometric analysis were performed with Quantity One software on a ChemiDoc XRS instrument (Bio-Rad Laboratories Inc.) and using β actin for normalization.
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