Total lysates proteins were extracted with cell lysis buffer (Beyotime, Shanghai, China) and were denatured by boiling. Next, the cell lysates were separated on 4–12% SDS- polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (PVDF Western Blotting Membranes, Roche). The membranes were blocked in PBS buffer [50 mM Tris–HCl, 150 mM NaCl, and 0.1% Tween (pH 7.6)] supplemented with 5% non-fat dry milk incubated with the appropriate antibodies for 12 h. An HRP-labeled secondary antibody and a chemiluminescent detection system (Phototope-HRP western blot detection kit; New England Biolabs, Ipswich, MA, United States) were used for developing the blots.
Phototope hrp western blot detection kit
Manufactured by New England Biolabs
Sourced in United States
The Phototope-HRP Western blot detection kit is a laboratory reagent used for the visualization and detection of target proteins in Western blot analysis. It contains the necessary components for the chemiluminescent detection of horseradish peroxidase (HRP)-conjugated secondary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf western blotting membrane, by Roche (2 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
Gs 700 densitometer, by Bio-Rad (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Sc 390351, by Santa Cruz Biotechnology (1 mentions)
Sc 374463, by Santa Cruz Biotechnology (1 mentions)
Hybond ecl membrane, by Cytiva (1 mentions)
Lumiglo chemiluminescent substrate, by Cell Signaling Technology (1 mentions)
Ift88, by Proteintech (1 mentions)
Broad spectrum protease inhibitor cocktail, by Roche (1 mentions)
Arl13b, by Proteintech (1 mentions)
Gapdh, by Abcam (1 mentions)
5 protocols using phototope hrp western blot detection kit
1
Western Blot Analysis of Protein Extracts
2
Protein Expression Analysis of CRIF1
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Western Blot was used to analyze the protein expression of CRIF1 in Jurkat cells and 40 BM samples. Total protein lysates were extracted with RIPA and denatured by boiling. Protein samples were resolved on 12% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Membranes were blocked in PBS containing 5% (w/v) nonfat dry milk and 0.1% Tween and incubated for 2 h with appropriate antibodies. Blots were developed using horseradish peroxidase-linked anti-mouse secondary antibody and developed with a chemiluminescent detection system (Phototope-HRP Western blot detection kit; New England Biolabs).
3
Quantitative Analysis of Bcl-2 and Bax Proteins
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Protein was extracted from frozen left ventricular tissue. Bovine serum albumin as a standard protein concentration was determined by the bicinchoninic acid protein assay (Pierce Rockford, IL). Equal proteins were loaded on 12% SDS-PAGE which were confirmed by Coomassie blue staining. The sample volume is 40 μg. Proteins were transferred to a polyvinylidene membrane [31 (link), 32 (link)]. Rat anti-Bcl-2 monoclonal antibody (Santa Cruz Biotechnology; Santa Cruz, CA) and mouse anti-Bax monoclonal antibody (Santa Cruz Biotechnology; Santa Cruz, CA) were used for Bcl-2 and Bax detection, respectively. The bands were visualized by Phototope-HRP Western blot detection kit (New England Biolabs, Beverly, MA) and were scanned by GS-700 densitometer (Bio-Rad Company, Hercules, CA) and then quantified by quantitative procedure. Optical density of the tissue samples was normalized to the control sample in arbitrary densitometry unit.
4
Western Blot Analysis of Protein Expression
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Protein expression in the samples was analysed by western blotting. The total protein lysate was extracted with cell lysis buffer for western blotting and immunoprecipitation (No. P0013, Beyotime) and denatured by boiling. Protein samples were resolved on 12 % SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (PVDF Western Blotting Membranes, Roche). Membranes were blocked in PBS containing 5 % (w/v) non-fat dry milk and 0.1 % Tween-20 for 2 h, incubated with the appropriate antibodies overnight, washed with TBST buffer, and then incubated with secondary antibodies for 2 h. Blots were developed using a horseradish peroxidase-linked secondary antibody and developed with a chemiluminescent detection system (Phototope-HRP Western blot detection kit, New England Biolab).
The primary antibodies used for blotting were as follows: anti-Smad5 (1:1000, 12534, Cell Signaling Technology), anti-p-Smad5 (1:1000, 9516S, Cell Signaling Technology), anti-OGN (1:1000, sc-374463, Santa Cruz), anti-Runx2 (1:1000, sc-390351; Santa Cruz), and anti-β-actin (1:1000, sc-47778, Santa Cruz).
The primary antibodies used for blotting were as follows: anti-Smad5 (1:1000, 12534, Cell Signaling Technology), anti-p-Smad5 (1:1000, 9516S, Cell Signaling Technology), anti-OGN (1:1000, sc-374463, Santa Cruz), anti-Runx2 (1:1000, sc-390351; Santa Cruz), and anti-β-actin (1:1000, sc-47778, Santa Cruz).
5
Western Blot Analysis of Cellular Proteins
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Cells were washed twice with PBS and lysed in RIPA lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 1% Nonidet P-40) supplemented with a broad-spectrum protease inhibitor cocktail (Roche). Protein concentrations were measured using the Bradford assay. Proteins were denatured by boiling for 5 min. Samples were resolved by 10% SDS-PAGE and transferred to Hybond ECL membranes (Amersham Pharmacia Biotech). The membranes were blocked for 30 min in Tris-buffered saline containing 0.1% Tween 20 (TBS/T) and 5% non-fat milk, and then incubated overnight at 4°C with primary antibodies against LC3 (Sigma-Aldrich), IFT88 (ProteinTech Group), Arl13B (ProteinTech Group), Polyglutamylation Modification (GT335, AdipoGen), and GAPDH (Abcam). The membranes were washed three times with TBS/T and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Phototope-HRP Western blot detection Kit; New England Biolabs) for 2 hr at room temperature. After three washes for 10 min each, the blots were developed using the LumiGLO chemiluminescent substrate (Cell Signaling Technology).
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