At 2, 4, 6, and 8 weeks (n = 3 per group at each time termination) after scaffold implantation, bone tissue specimens were homogenized in RIPA (Bio-Rad, Hercules, CA, USA) supplemented with protease inhibitor. After incubation at 4 °C for 30 min, the specimens underwent centrifugation at 14,000 rpm for 15 min. Protein concentration was measured with a BCA protein assay kit (Thermo, Rockford, MD, USA). Protein samples were subjected to 10% (w/v) SDS-PAGE, and separated proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking with 7% skim milk, the membranes were incubated overnight at 4 °C with primary antibodies against BMP2 and VEGFA (1:2000; Abcam, Cambridge, UK), and then incubated with secondary antibody (Cell Signaling Technology®, Beverly, MA, USA) at room temperature for 1 h. The bands were captured using an imaging system (ImageQuant™ LAS 4000 mini, GE Healthcare Life Sciences, Chicago, USA), and then semi-quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Radioimmunoprecipitation assay (ripa)
Manufactured by Bio-Rad
Sourced in United States
RIPA is a buffer solution used for cell lysis and protein extraction in biological research. It contains a combination of detergents and other agents that help solubilize and extract proteins from cells and tissues, while maintaining their native structure and function. RIPA is commonly used in various biochemical and molecular biology techniques, such as Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assays (ELISA).
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Imagequant las 4000 mini, by GE Healthcare (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ab97592, by Abcam (1 mentions)
Ab37168, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Mbs435036, by MyBioSource (1 mentions)
Anti laminin l9393, by Merck Group (1 mentions)
Anti gas2 ab55076 100, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by PerkinElmer (1 mentions)
4 protocols using radioimmunoprecipitation assay (ripa)
1
Quantifying Bone Regeneration Biomarkers
2
Quantifying ROCK1 Expression in lncRNA CCHE1 Knockdown
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Knockdown rate of lncRNA CCHE1 were reached before this experiment. RIPA (Bio-Rad) was used to isolate proteins. Proteins were separated using 12% SDS-PAGE gel. Western blotting was performed using conventional method. Primary antibodies included rabbit anti-human ROCK1 (1: 1200, ab97592, Abcam) and rabbit anti-human GAPDH antibody (1: 1200, ab37168, Abcam). IgG-HRP (1:1000, MBS435036, MyBioSource) was used as the secondary antibody. ECL (Sigma-Aldrich, USA) was used for signal development and data normalization was performed using Image J software.
3
Western Blot Analysis of GAS2 and Laminin
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Tissue samples were homogenized and lysed in RIPA (Bio-Rad Laboratories, Hercules, CA, USA) buffer, run on a 12% polyacrylamide gel, and wet transferred to Bio-Rad PVDF membrane (Bio-Rad Laboratories) according to the standard protocol and chemiluminescently visualized using HRP-conjugated secondary antibodies from PerkinElmer (Waltham, MA, USA). Anti-GAS2 (ab55076-100) from Abcam (Cambridge, MA, USA) and anti-laminin (L9393) from Sigma-Aldrich (St. Louis, MO, USA).
4
Quantifying Spinal Dural Adhesion Proteins
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Adhesion tissues behind the spinal dural in different groups were collected and homogenized in 300 mL RIPA (Bio-Rad Laboratories Inc., Hercules, CA, USA). The mixed lysates were stewing on ice for 30 minutes and centrifuged at 12,000 rpm in a refrigerated centrifuge for 15 minutes to extract the supernatant. The protein concentrations of the supernatant were determined by a BCA protein assay kit (Thermo Fisher Scientific). The samples were electrophoresed through 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto poly-vinylidene fluoride (PVDF) membranes. Afterward, the membranes were incubated with antibodies (diluted 1:1,000, Abcam, Cambridge, MA, USA) against β-actin, collagen I (col I), collagen III (col III), α-SMA and TGF-β/SMAD2/3 at 4°C overnight, followed by incubation with the secondary antibodies (diluted 1:2,000; ZSGB-BIO, Beijing, China) at 37°C for 1 hour. Finally, the membranes were washed with TBST buffer for three times, and the protein bands were scanned with an imaging system (Image Quant LAS 4000 mini, GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
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