Whole-cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM MgCl2) supplemented with protease inhibitors (Roche). Protein samples were separated by SDS-PAGE and then transferred to cellulose nitrate membranes. After incubation in 5% nonfat milk for 30 min, the membrane was incubated with the primary antibody overnight at 4°C and then with the horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The target proteins were detected with the Luminata Crescendo immunoblotting HRP substrate (Millipore, Billerica, MA). Each experiment was repeated at least three times using separate batches of cells.
Luminata crescendo immunoblotting hrp substrate
Manufactured by Merck Group
Luminata Crescendo immunoblotting HRP substrate is a chemiluminescent reagent that is used to detect and quantify proteins in Western blot analysis. It is designed to produce a high-intensity, sustained luminescent signal when reacted with horseradish peroxidase (HRP) conjugated to antibodies.
Automatically generated - may contain errors
4 protocols using luminata crescendo immunoblotting hrp substrate
1
Western Blot Analysis of Proteins
2
Western Blot Protein Detection Protocol
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Whole-cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin and other inhibitors) supplemented with protease inhibitors (Roche). Protein samples were separated by SDS-PAGE and subsequently transferred to cellulose nitrate membranes. The membrane was incubated in 5% nonfat milk for 1 h and then washed with TBST four times at room temperature for 10 min for the first two times followed by 5 min for the third and fourth times. The membrane was incubated with the primary antibody at 4°C overnight, and then washed four times with TBST at room temperature for 10 min for the first two times followed by 5 min for the third and fourth times. The membrane was then incubated with the second antibody for 1 h at room temperature. The target proteins were detected with the Luminata Crescendo immunoblotting HRP substrate (Millipore, Billerica, MA).
3
Protein Extraction and Western Blot Analysis
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The cells were lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and 2 mM MgCl2) supplemented with a protease and phosphatase inhibitor cocktail (HY-K0010 and HY-K0022, MedChemExpress). Protein samples were separated by SDS-PAGE and then transferred to polypropylene fluoride membranes (C3117, Millipore, MA, USA). After incubation in 5% nonfat milk (A600669, Sangon Biotech) for 30 min, the membrane was incubated with the primary antibody overnight at 4°C and then with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. The target proteins were detected with the Luminata Crescendo immunoblotting HRP substrate (WBLUR0500, Millipore) on a GE AI600 imaging system.
4
Protein Extraction and Western Blot Analysis
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The cells were lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and 2 mM MgCl2) supplemented with a protease and phosphatase inhibitor cocktail (HY-K0010 and HY-K0022, MedChemExpress). Protein samples were separated by SDS-PAGE and then transferred to polypropylene fluoride membranes (C3117, Millipore, MA, USA). After incubation in 5% nonfat milk (A600669, Sangon Biotech) for 30 min, the membrane was incubated with the primary antibody overnight at 4°C and then with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. The target proteins were detected with the Luminata Crescendo immunoblotting HRP substrate (WBLUR0500, Millipore) on a GE AI600 imaging system.
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