Collagen/hydroxyapatite composites were prepared according to a previously established protocol [33 (link)]. Briefly, the required amount of hydroxyapatite nanoparticles (10 µm powder, Fluidinova, Moreira da Maia, Portugal) was weighted, washed with TBS (tris buffered saline, Biolegend), and then equilibrated in TBS overnight. Thereafter, the TBS/HA solution was centrifuged for 20 min at 3000 rpm. TBS supernatant was discarded and a VEGF-mimicry peptide solution (200 µg peptide in 400 µL TBS (molar concentration: QK—0.52 mM; E7-QK—0.35 mM) per 10 mg HA) was added and incubated with HA for 2 h while rotating (15 rpm). The respective volume of the bovine type I collagen solution (HA: Coll = 2:1) (Advanced Biomatrix, San Diego, CA, USA) was added and then put into an incubator for polymerization at 37 °C for 30 min.
Tris buffered saline (tbs)
Manufactured by BioLegend
Sourced in Portugal, United States
Tris buffered saline is a solution composed of Tris (hydroxymethyl) aminomethane and sodium chloride. It is commonly used as a buffer in various biological and biochemical applications to maintain a stable pH environment.
Automatically generated - may contain errors
Lab products found in correlation
Type 1 bovine collagen solution, by Advanced BioMatrix (1 mentions)
Ab194519, by Abcam (1 mentions)
Ab124957, by Abcam (1 mentions)
Chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Ab92333, by Abcam (1 mentions)
Bca kit, by Boster Bio (1 mentions)
Ab7090, by Abcam (1 mentions)
Anti nf κb 8242, by Cell Signaling Technology (1 mentions)
Ab8227, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Ab183559, by Abcam (1 mentions)
2 protocols using tris buffered saline (tbs)
1
Biomimetic Collagen-Hydroxyapatite Composites
2
Western Blot Analysis of Protein Markers
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Total protein content was assessed using a bicinchoninic acid (BCA) kit (Bosterbio, Pleasanton, USA). An equal amount of protein was used in SDS-PAGE. Proteins were transferred onto the polyvinylidene fluoride (PVDF) membranes (Biocompare, San Francisco, USA) after electrophoresis, followed by the blocking with 5% skim milk powder in Tween and Tris-buffered saline (BioLegend, San Diego, USA) for 50 min. These membranes were incubated with primary antibodies including anti-Capn4 (ab92333; 1:2000; Abcam, Cambridge, USA), anti-NF-κB (#8242; 1:1000; Cell Signaling, Beijing, China), anti-phospho-NF-κB (ab183559; 1:1000; Abcam), anti-IKK-β (ab124957; 1:1000; Abcam), anti-p-IKK-β (ab194519; 1:1000; Abcam), and β-actin (ab8227; 1:2000; Abcam) at 4°C overnight followed by the incubation with Goat anti-Rabbit horseradish peroxidase (HRP) (ab7090; 1:2000; Abcam) at 37°C for 1 h. β-actin was the internal control. The chemiluminescence reagent (Thermo Fisher Scientific) was used to visualize the bands and detected under a Las-3000 imaging system (Biocompare).
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