Protein extracts from cells were quantified by BCA (Pierce), separated by SDS-PAGE (10% acrylamide), transferred to nitrocellulose membrane (0.2 µm, Bio-Rad), labeled with primary antibodies (1–5 µg/ml), washed, and labeled with species-specific HRP-conjugated secondary antibodies (Thermo Fisher Scientific). Peroxidase activity was detected using Western Lightning Chemiluminescence Reagent (GE Healthcare). The molecular migration position of transferred proteins was compared with the PageRuler Prestain Protein Ladder 10–170 kD (Thermo Fisher Scientific). Signal quantification was performed using ImageJ.
Species specific hrp conjugated secondary antibodies
Manufactured by Thermo Fisher Scientific
Sourced in Spain, United States
Species-specific HRP-conjugated secondary antibodies are laboratory reagents that bind to primary antibodies from a specific animal species. The HRP (horseradish peroxidase) conjugate enables detection and visualization of the target molecules in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
0.2 m nitrocellulose membrane, by Bio-Rad (1 mentions)
Western lightning chemiluminescence reagent, by GE Healthcare (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Cell lytic, by Merck Group (1 mentions)
Acrylamide, by Bio-Rad (1 mentions)
Bradford s reagent, by Bio-Rad (1 mentions)
Tris glycine sds buffer 10, by Bio-Rad (1 mentions)
Tris glycine buffer 10, by Bio-Rad (1 mentions)
5 bromo 2 deoxyuridine brdu cell proliferation assay kit, by Abcam (1 mentions)
Tween 20, by Merck Group (1 mentions)
Collagenase, by Worthington (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
C digit chemiluminescence western blot scanner, by LI COR (1 mentions)
Complete protease inhibitor tablet, by Roche (1 mentions)
4 protocols using species specific hrp conjugated secondary antibodies
1
Western Blot Protein Quantification
2
Collagenase-based Cell Extraction Protocol
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Collagenase was obtained from Worthington Biochemical Corporation (Labclinics, Madrid, Spain). Cell Lytic for cell lysis and protein solubilization, crystal violet, protease inhibitor cocktail (Complete, EDTA-free), thapsigargin and Tween-20 were purchased from Sigma Chemicals Co. (Madrid, Spain). Fetal bovine serum, Hank’s balanced salts (HBSS), horse serum, medium 199 and SuperSignal West Femto were obtained from Fisher Scientific Inc. (Madrid, Spain). Polystyrene plates for cell culture were obtained from Thermo Fisher Sci. (Madrid, Spain). Penicillin/streptomycin was purchased from BioWhittaker (Lonza, Basel, Switzerland). Acrylamide, Bradford´s reagent, Tris/glycine/SDS buffer (10×) and Tris/glycine buffer (10×) were from Bio-Rad (Madrid, Spain). 5-Bromo-2-deoxyuridine (BrdU) cell proliferation assay kit was purchased from BioVision (Deltaclon S.L., Madrid, Spain). SB203580 and U0126 were obtained from Tocris (Biogen Científica, Madrid, Spain). Species-specific HRP-conjugated secondary antibodies were purchased from Thermo Fisher Sci. (Madrid, Spain). All other analytical-grade chemicals used were obtained from Sigma Chemicals Co. (Madrid, Spain).
3
Western Blot Analysis of Protein Extracts
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Cells were lysed in SDS buffer containing protease and phosphatase inhibitor cocktails (Thermo Fisher, USA). BCA protein assay kit (Thermo Fisher, USA) was used to measure the protein concentration of the lysate. After normalization, protein extracts were separated by 8%, 10%, 12%, or 15% SDS-PAGE and transferred onto the PVDF membrane (Bio-Rad Laboratories, USA); the membranes were then incubated with primary antibodies at 4°C over night. The next day, blots were incubated with species-specific HRP-conjugated secondary antibodies (Thermo Fisher, USA). Signal quantification was performed by enhanced chemiluminescence (ECL, Pierce).
4
Quantifying Intracellular Protein Levels in siRNA Transfected Cells
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To assess intracellular protein levels in siRNA transfected cells, forty-eight hours posttransfection cells were lysed on ice with lysis buffer (0.5M HEPES, 1.25M NaCl, 1M MgCl 2 , 0.25M EDTA, 0.1% Triton X-100, 1X complete protease inhibitor tablets (Roche, Indianapolis, IN)). and centrifuged at 4°C for 20 minutes Supernatants were collected and boiled for 10 minutes after addition of 5X SDS-PAGE sample buffer (5X Sample Buffer; 0.312M Tris pH 6.8, 25% 2-Mercaptoethanol, 50% glycerol and 10% SDS). Samples were run on a 14% SDS-PAGE gel and transferred onto a nitrocellulose membrane. Membranes were blocked in 5% skim milk powder in TBST for 1 hour, and incubated overnight at 4°C with antibodies specific for the ACTH peptide residues 1-18 (1:1000; provided by Dr. Iris Lindberg, University of Maryland) or actin (1:3000; Thermo Scientific). Membranes were washed 3X in TBST, incubated in speciesspecific HRP-conjugated secondary antibodies for 2 hours (1:2000; Thermo Scientific), and washed 3X in TBST. All blots were imaged using the C-Digit chemiluminescence Western blot scanner (LI-COR Biosciences, Lincoln, NE) with Crescendo ECL substrate (Millipore Inc.; Billerica, MA). Band intensities corresponding to peptides of 29 kDa, 26 kDa, and 9 kDa were quantified using the ImageQuant Studio software (LI-COR) and normalized to the actin loading control.
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