Cells were lysed with RIPA buffer (Cell Signaling Technology, Inc.). Following centrifugation at 4°C for 20 min at 12,000 × g, the supernatant of the cell lysate was incubated with a primary antibody overnight at 4°C. Subsequently, the supernatant was incubated with 50 µl protein A beads (GE Healthcare) for 4 h at 4°C, and the beads were washed with RIPA buffer three times. The immunoprecipitated proteins were mixed with Laemmli (Cell Signaling Technology, Inc.) buffer and boiled for 5 min at 100°C prior to western blot analysis as aforementioned.
Ripa buffer
Manufactured by GE Healthcare
Sourced in United States
RIPA buffer is a commonly used laboratory reagent designed for protein extraction and solubilization. It is a mixture of various detergents and salts that help to disrupt cell membranes and release proteins from cells. The buffer is typically used in immunoprecipitation, Western blotting, and other protein analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescence detection kit, by Merck Group (2 mentions)
Phosphatase inhibitor, by Merck Group (2 mentions)
Chemidoc system, by Bio-Rad (2 mentions)
Protein a beads, by GE Healthcare (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by GE Healthcare (1 mentions)
Protease inhibitor, by Fujifilm (1 mentions)
Sample grinding kit, by GE Healthcare (1 mentions)
Ripa buffer, by Fujifilm (1 mentions)
4 protocols using ripa buffer
1
Protein Immunoprecipitation and Western Blot Analysis
2
Protein Extraction from Tumor Cells
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Cell pellets were mechanically broken down in RIPA buffer (Wako, Osaka, Japan), which contained protease inhibitor (Wako), and centrifuged at 15,000 rpm for 5 min at 4 °C. Supernatants were collected and analyzed with the immuno-wall assay. In order to lyse the tumor tissues, the tissues were placed in 1.5 ml tubes containing 200 μl RIPA buffer, a protease inhibitor, and resin beads, which were then collectively ground using pestles from a sample-grinding kit (GE Healthcare, Little Chalfont, UK). The lysate was then centrifuged at 15,000 rpm for 5 min at 4 °C and the supernatants were collected and analyzed. Approximately 100 μg of protein was extracted from 105 cells.
3
Quantifying Cellular Amyloid Processing
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Cells were treated with miRNA for 24 h and collected to evaluate the expression of amyloid precursor protein (APP) and its C-terminal fragments (CTFs). To prepare whole-cell lysates, cells were washed with ice-cold PBS, scraped into radioimmunoprecipitation assay (RIPA) buffer (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) containing a cocktail of both protease inhibitors and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA), sonicated to homogenize the cell suspension, and centrifuged to remove cell debris. Total protein extract (10-100 µg) was subjected to western blot analysis with an appropriate antibody. The isolated protein was electrophoresed in a 10% or 15% polyacrylamide-SDS gel and transferred to a nitrocellulose membrane. After blocking for 1 h with 5% skim milk, the membrane was incubated with a primary antibody. After washing, the membrane was then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG antibody. Immunoreactive signals were detected with an enhanced chemiluminescence detection kit (Merck Millipore, Temecula, CA, USA) and analyzed using a Chemi-Doc System (Bio-Rad).
4
Quantifying Cellular Amyloid Processing
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Cells were treated with miRNA for 24 h and collected to evaluate the expression of amyloid precursor protein (APP) and its C-terminal fragments (CTFs). To prepare whole-cell lysates, cells were washed with ice-cold PBS, scraped into radioimmunoprecipitation assay (RIPA) buffer (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) containing a cocktail of both protease inhibitors and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA), sonicated to homogenize the cell suspension, and centrifuged to remove cell debris. Total protein extract (10-100 µg) was subjected to western blot analysis with an appropriate antibody. The isolated protein was electrophoresed in a 10% or 15% polyacrylamide-SDS gel and transferred to a nitrocellulose membrane. After blocking for 1 h with 5% skim milk, the membrane was incubated with a primary antibody. After washing, the membrane was then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG antibody. Immunoreactive signals were detected with an enhanced chemiluminescence detection kit (Merck Millipore, Temecula, CA, USA) and analyzed using a Chemi-Doc System (Bio-Rad).
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