To measure PFN, α-tubulin levels in cells, cells were collected 12–18 h after transfection and incubated for 30 min at 4°C in lysis buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 7.5, 2 mM EDTA, 0.2 mM sodium orthovanadate, 20 mM β-glycerophosphate, 50 mM sodium fluoride, 1 mM PMSF, 1 mM DTT, and 1× Roche complete protease inhibitor mixture). Samples were precleared by centrifugation at 16,500 × g for 30 min at 4°C, quantified by Bradford assay, and immunoblotted. Blots were probed with 1:1000 primary antibody ab50667 against Profilin1 (Abcam, Cambridge, UK) or 1:2000 primary antibody sc-32292 against α-tubulin (Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 2h. Blots were then probed with secondary horseradish peroxidase antibodies (GE Healthcare), and proteins were detected using Pierce ECL Western Blotting Substrate detection kit (Thermo Fisher). Bands were quantified by densitometry using Fiji.
Pierce ecl western blotting substrate detection kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce ECL Western Blotting Substrate detection kit is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. The kit provides the necessary components for the visualization of target proteins labeled with HRP-conjugated antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Secondary horseradish peroxidase antibodies, by GE Healthcare (2 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Ab15270, by Abcam (1 mentions)
Ab6556, by Abcam (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Trans blot membrane, by Bio-Rad (1 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
5 protocols using pierce ecl western blotting substrate detection kit
1
Quantifying Profilin1 and α-Tubulin Levels
2
Protein Extraction and Immunoblotting Protocol
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Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-base pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid (EDTA) supplemented with protease and phosphatase inhibitors (Roche)). After 15 min of lysis on ice, the cells were centrifuged at 14,000 rpm for 15 min at 4 °C to collect the supernatants for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Protein concentrations were determined with Bio-Rad Bradford Protein Assay (Bio-Rad) using bovine serum albumin (BSA) as a standard. Protein bands recognized by specific antibodies were visualized using Pierce ECL Western Blotting Substrate detection kit (Thermo Fisher Scientific). Densitometric analysis was performed using Image J software (NIH).
3
Quantifying Protein Levels in Silenced Cells
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To measure APC levels in cells after silencing and rescue, cells were collected 48 h after initial oligo transfection and incubated for 30 min at 4°C in lysis buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 7.5, 2 mM EDTA, 0.2 mM sodium orthovanadate, 20 mM β-glycerolphosphate, 50 mM sodium fluoride, 1 mM PMSF, 1 mM DTT, and 1× Roche complete protease inhibitor mixture). Samples were precleared by centrifugation at 14,000 rpm for 30 min at 4°C, quantified by Bradford assay, and immunoblotted. For measuring phospho-Paxillin, total Paxillin, and phospho–FAK-pY397 levels, the same procedure was used except that cells were incubated in serum-free media overnight before collection. Blots were probed with 1:500 rabbit anti-APC (ab15270; Abcam), 1:2,000 rabbit anti–GFP (ab6556; Abcam), 1:500 rabbit anti–phospho-Paxillin (Tyr118; PP4501; ECM Biosciences), 1:1,000 rabbit anti–FAK-phospho–tyrosine 397 (FAK-pY397; 141–9; Invitrogen), 1:1,000 mouse/human anti-Paxillin (AHO0492; Invitrogen), or 1:2,000 mouse anti–α-tubulin antibody (sc-32292; Santa Cruz Biotechnology). Blots were then probed with secondary horseradish peroxidase antibodies (GE Healthcare), and proteins were detected using a Pierce ECL Western Blotting Substrate detection kit (Thermo Fisher Scientific). Bands were quantified using Fiji.
4
Quantifying Protein Levels via Western Blot
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To measure protein levels in cells after silencing and rescue, cells were harvest 48 hr after initial oligo transfection and incubated for 10 min at 4°C in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 2 mM EDTA, 50 mM NaF). Samples were incubated on ice for 30 min, vortexed every 10 min, then precleared by centrifugation at 20,800 x g for 15 min at 4°C, quantified by Bradford assay, and immunoblotted. Proteins were detected using a Pierce ECL Western Blotting Substrate detection kit (Thermo Fisher Scientific). Bands were quantified using ImageLab (Biorad).
5
Western Blotting Protocol for Testicular Proteins
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Testes were homogenated using a Tissue‐Tearor (RPI Corp, Mt. Prospect, IL, USA) in lysis buffer containing 50 mmol/L Tris‐HCl, 150 mmol/L NaCl, 1% NP‐40, 1 mmol/L EDTA, 2 mmol/L MgCl2, 0.5% sodium deoxycholate, complete protease inhibitor and phosphorylase inhibitor cocktail (Roche, Mannheim, Germany). The protein concentrations were determined using Bradford protein assay kits (Bio‐Rad, Hercules, CA, USA). 20 μg testicular homogenates each sample was electrophoresed in 10% SDS/PAGE gels and then electroblotted to TransBlot membranes (Bio‐Rad). After blocking with 3% non‐fat milk in Tris‐buffered Saline‐Tween‐20, membranes were incubated with the primary antibody as listed in Table 2 , followed by horseradish peroxidase (HRP)‐conjugated secondary antibodies (Santa Cruz Biotech). Signal was detected using a Pierce ECL Western blotting substrate detection kit (Thermo Fisher Scientific, Waltham, MA, USA). All membranes were reblotted with β‐actin or glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody as the loading control. The intensity of specific bands was scanned using image analysis software Total Lab. The results were presented as the ratio of target protein over β‐actin or GAPDH.
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