The whole-cell lysate was prepared using Radioimmunoprecipitation assay (RIPA) lysis buffer (25mM Tris–HCl pH 7.6, 150mM NaCl, 1% Triton, and 0.1% SDS) + protease inhibitor and phosphatase inhibitor cocktail. Protein concentrations were estimated using a Bradford Assay (Bio-Rad Catalog # 500–0006). Lysates (25 μg) were mixed with 6X loading dye containing b-ME followed by boiling at 95°C on a dry bath, then loaded. The gel was transferred to a nitrocellulose membrane. The membrane was blocked with 5% BSA for 1 hour at room temperature. Primary antibodies were prepared in a 5% BSA solution as well and the membranes were incubated overnight at 4°C with gentle shaking. Antibodies used to detect the target proteins: MCU (Santa Cruz), MCUR1 (Santa Cruz), Calcineurin (Cell Signaling), Drp1 (Novus), phosphor-Drp1S637 (Cell Signaling), Fis1 (Santa Cruz), and NMDAR (Cell Signaling). Species-specific secondary antibodies were used from Santa Cruz and the membranes were incubated for 1 hour at room temperature. Chemiluminescence was used to detect the signal. The densitometry ratio of the bands was determined using an ImageJ that was normalized to GAPDH.
Phosphor drp1s637
Manufactured by Cell Signaling Technology
Phosphor-Drp1S637 is a product offered by Cell Signaling Technology. It is a laboratory reagent used for the detection and quantification of phosphorylation of the serine 637 residue of the Dynamin-related protein 1 (Drp1).
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using phosphor drp1s637
1
Whole-Cell Lysate Preparation and Western Blot Analysis
2
Quantifying Mitochondrial Regulators by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The whole-cell lysate was prepared using Radioimmunoprecipitation assay (RIPA) lysis buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% Triton, and 0.1% SDS) + protease inhibitor and phosphatase inhibitor cocktail. Protein concentrations were estimated using a Bradford Assay (Bio-Rad Catalog # 500-0006). Lysates (25 μg) were mixed with 6X loading dye containing b-ME followed by boiling at 95°C on a dry bath, then loaded. The gel was transferred to a nitrocellulose membrane. The membrane was blocked with 5% BSA for 1 hour at room temperature. Primary antibodies were prepared in a 5% BSA solution as well and the membranes were incubated overnight at 4°C with gentle shaking. Antibodies used to detect the target proteins: MCU (Santa Cruz), MCUR1 (Santa Cruz), Calcineurin (Cell Signaling), Drp1 (Novus), phosphor-Drp1S637 (Cell Signaling), Fis1 (Santa Cruz), and NMDAR (Cell Signaling). Species-specific secondary antibodies were used from Santa Cruz and the membranes were incubated for 1 hour at room temperature. Chemiluminescence was used to detect the signal. The densitometry ratio of the bands was determined using an ImageJ that was normalized to GAPDH.
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