Western blot analyses were carried out as described previously [4 (link), 28 (link)]. Briefly, LPCs were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (P0013D, Beyotime) supplemented with protease inhibitors (Roche, Basel, Switzerland) and phosphatase inhibitors (PhoSSTOP Cocktail Tablets, Roche) on ice, after which the concentration of the proteins was measured via BCA protein assay kits (Beyotime). Equal amounts of cell lysate were then mixed with sodium dodecyl sulfate (SDS) loading buffer, denatured, loaded and separated via SDS–polyacrylamide (SDS–PAGE) gel (Bio-Rad, Bio-Rad, Hercules, CA, USA). After separation, the proteins were transferred to PVDF membranes (Roche), after which the proteins were incubated with primary antibodies overnight at 4°C. After incubation, the membrane was washed with TBST 3 times (5 min each) and then incubated for 1 h with a horseradish peroxidase (HRP)-labeled secondary antibody at 37°C. Finally, the membrane was washed again in TBST 3 times, reactive bands on the membrane were detected by enhanced chemiluminescence (ECL) reagent (Pierce Protein Biology, Thermo Fisher Scientific, Waltham, MA, USA), and the signals were visualized by an image labeling imaging system (Bio-Rad).
Phosstop cocktail tablets
Manufactured by Roche
Sourced in Switzerland, Italy
PhosSTOP Cocktail Tablets are a product designed for phosphatase inhibition in biological sample preparation. The tablets contain a combination of phosphatase inhibitors that can be used to prevent the dephosphorylation of proteins during sample lysis and processing.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay reagent, by Bio-Rad (2 mentions)
Complete protease inhibitor cocktail tablet, by Roche (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Roche (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Immobilon pvdf membrane, by Merck Group (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Complete mini, by Roche (1 mentions)
Complete ultra, by Roche (1 mentions)
Mouse anti β actin, by Abcam (1 mentions)
Hrp conjugated goat anti rabbit igg antibody, by Cell Signaling Technology (1 mentions)
Gradient gel, by Bio-Rad (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
6 protocols using phosstop cocktail tablets
1
Western Blot Analysis Protocol
2
Protein Extraction from Larval Pellets
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Larval pellets (50–100 mg) were homogenized in two volumes of RIPA lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.6, 5 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (1 mM PMSF and Complete Protease Inhibitor Cocktail Tablets, Roche) and phosphatase inhibitors (PhosSTOP Cocktail Tablets, Roche). After centrifugation, protein concentration was determined using a Bio-Rad Protein Assay Reagent (Bio-Rad) with bovine serum albumin as a standard.
3
Protein Extraction from Larval Pellets
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Larval pellets were homogenized in two volumes of RIPA lysis buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.6, 5 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (1 mM PMSF and Complete Protease Inhibitor Cocktail Tablets, Roche, Monza, Italy) and phosphatase inhibitors (PhosSTOP Cocktail Tablets, Roche). After centrifugation, protein concentration was determined using a Bio-Rad protein assay reagent (Bio-Rad, Milan, Italy) with bovine serum albumin as a standard.
4
Western Blot Analysis of Renal Proteins
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A total of 150 mg of renal cortex tissue were homogenized (PT10/35GT homogenizer Polytron®; Kinematica AG), in cold lysis buffer (1 mM DTT, 10 mM Tris-HCl, 1 mM EDTA, 1 mM sodium orthovanadate, 15 mM sodium azide, 1.0% Triton X-100 and 30% glycerol) supplemented with the commercial protease inhibitor cOmplete™ Mini and phosphatase inhibitor PhosSTOP™ Cocktail tablets (Roche Diagnostics GmbH). After homogenates' centrifugation (13,000 × g for 30 min at 4°C) (Thermo Fisher Scientific, Inc.; Legend RT+ centrifuge), supernatants were recovered and total protein was quantified by the Bradford method (Bio-Rad Laboratories Inc.). Equal protein amounts (60–80 µg) were electrophoresed in a 10% SDS-PAGE polyacrylamide gel and transferred to an Immobilon® PVDF membrane (EMD Millipore). Membranes were blocked for 1 h at room temperature with 1% nonfat dry milk and incubated with the primary antibodies against p65 (1:250), IκBα (1:250), EGFR (1:250) or α-tubulin (1:1,000) overnight at 4°C. Subsequently, membranes were washed and incubated with the corresponding secondary antibody (1:30,000 for anti-rabbit and anti-mouse). Immunoreactive signal bands were detected using Immobilon™ Western Chemiluminescent HRP Substrate (EMD Millipore) and were recorded on X-ray films and quantified by densitometry, as described below.
5
Western Blot Analysis of Bone Marrow Stromal Cells
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BMSCs were scraped in RIPA lysis buffer containing cOmplete protease inhibitor and PhosSTOP cocktail tablets (Roche). Following marrow flush, diaphyseal bone was chopped with scissors 100 times in RIPA (50 mM Tris (pH 7.4), 15 mM NaCl, 0.5% NP-40, 0.1% SDS, 0.1% sodium deoxycholate) lysis buffer. Protein concentration was determined by BCA protein assay kit (Thermo). Protein (20 g) was loaded on 12% polyacrylamide gel and transferred onto Imuno-Blot PVDF membrane. The membranes were blocked for 1 h at room temperature in 5% milk powder in TBS with 0.1% Tween (TBST) and then incubated at 4°C with the primary antibody overnight. Primary antibodies were used at 1:1000 to detect proteins, listed as follows: Anti-SLC38A2 (RRID:AB_2050321), Anti-Runx2 (RRID:AB_10949892), Anti-ACTB (RRID:AB_330288), Anti-ATUB (RRID:AB_2619646), Anti-Phospho T172 AMPK (RRID:AB_330330) and Anti-AMPK (RRID:AB_330331). Membranes were then incubated at room temperature with Anti-Rabbit IgG (RRID:AB_2099233) or Anti-Mouse IgG, HRP-linked Antibody (RRID:AB_330924) at 1:2000 for 1 h at room temperature. Immunoblots were next developed by enhanced chemiluminescence (Clarity Substrate Kit or SuperSignal West Femto substrate). Each experiment was repeated with at least three independently prepared protein extractions. Densitometry was performed for quantification for each blot.
6
Western Blotting of Phosphorylated STAT6
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