For analysis of cytosolic proteins, cells were lysed in cell lysis buffer [50 mm HEPES (pH 7.5); 1 mm DTT, 150 mM NaCl, 1 mM EDTA, 0.1 % Tween 20, 10 % glycerol, 10 mm β-glycerophosphate, 1 mM NaF, 0.1 mm orthovanadate, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 0.1 mM PMSF] and were incubated at 4 °C for 30 min. Protein concentration was measured using Bio-Rad protein assay dye concentrate. Lysates (30 μg) were resolved electrophoretically on 10 % SDS-polyacrylamide gel and electrotransferred to a polyvinylidine difluoride membrane (Bio-Rad) using a tank blot procedure (Bio-Rad Mini Protean II). The membranes were incubated with the following primary antibodies: Heme oxygenase-1 (HO-1) (Santa Cruz, Cat # sc-10789), cleaved caspase-3 (Cell Signaling, Cat # 9661), Bax (Santa Cruz Biotechnologies, Cat # 20067), Bcl2 (BD-Transduction Laboratories, Cat # 551052), β-actin (Cell Signaling Technologies, Cat # 4967), NOX4 (Abcam, Cat # ab60940), Mn-SOD (Abcam, Cat # 13533), Cu/Zn SOD (Abcam, Cat #13498), COX IV (Abcam, ab14744) and 1:1000 dilutions of respective horseradish peroxidase-linked F(ab) fragment secondary antibody (Amersham Corp., Piscataway, NJ) for 1 h. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL) detection system (Amersham) as described previously [20 (link), 22 (link)].
Protein assay dye concentrate
Manufactured by Bio-Rad
Sourced in United States
The Protein assay dye concentrate is a solution used for the quantitative determination of protein concentration in a sample. It provides a colorimetric reaction that can be measured using a spectrophotometer.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Polyvinylidine difluoride membrane, by Bio-Rad (1 mentions)
Mini protean 2, by Bio-Rad (1 mentions)
Cox 4, by Abcam (1 mentions)
Ecl detection system, by Cytiva (1 mentions)
Heme oxygenase 1 ho 1, by Santa Cruz Biotechnology (1 mentions)
Mnsod, by Abcam (1 mentions)
Cuznsod, by Abcam (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Ab109374, by Merck Group (1 mentions)
P0448, by Agilent Technologies (1 mentions)
0.2 μm polyvinylidene difluoride membranes, by Bio-Rad (1 mentions)
Polyclonal goat anti mouse, by Agilent Technologies (1 mentions)
Superblock blocking buffer, by Thermo Fisher Scientific (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
7 protocols using protein assay dye concentrate
1
Cytosolic Protein Analysis Protocol
2
Western Blot Analysis of UCP1 Protein
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A third portion of collected BAT was homogenized in lysis buffer containing protease inhibitor cocktail tablets (Roche Diagnostics), and the protein concentration was determined using the Bradford method (Protein assay dye concentrate, Bio-Rad Laboratories).Ten micrograms of total protein were subjected to SDS-PAGE on 10% polyacrylamide gels, and electro-transferred to polyvinylidene difluoride membranes with a semidry blotter. Membranes were blocked in TBS/Tween with 3% of BSA (Bovine serum albumin, Sigma Aldrich) and probed with antibodies against UCP1 (Abcam) and a-tubulin (Sigma-Aldrich). Membranes were incubated with the corresponding secondary antibody: anti-rabbit for UCP1 and anti-mouse for a-tubulin (DAKO). Detection of proteins was performed with Enhanced chemiluminiscence (ECL) reagents (Pierce ECL Western Blotting Substrate, Cultek) according to the manufacturer’s instructions, exposed to x-ray films, developed and fixed under appropriate dark room conditions. Autoradiographic films were scanned and the band signal were quantified by densitometry using ImageJ-1.33 software (NIH), as formerly shown29 (link)–31 (link). Values were expressed in relation to a-tubulin. Adobe PhotoShop CC were used to crop and assemble the images for the presentations.
3
Western Blot Analysis of HMGA Proteins
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Whole cell lysates were prepared by re-suspending cells in ice-cold RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1%Triton X-100, 0.1% Sodium deoxycholate, 0.1% SDS and 140 mM NaCl) containing Protease Inhibitor cocktail (Roche) and sonicated on ice (5 W, 10x 3s). Protein concentration was determined using Bio-Rad Protein Assay Dye Concentrate. Samples were heated at 95°C for 10 min and separated by electrophoresis, transferred onto 0.2 μm polyvinylidene difluoride membranes (Bio-Rad) and blocked in superblock blocking buffer (Thermo Fisher Scientific). Membranes were incubated with primary antibodies (α-HMGA2 (CST 5269; 1:1000; RRID:AB_10694917, Ab41878; 1:200; RRID:AB_2279684), α-HMGA1 (CST 7777S; 1:1000; D6A4), α-Topoisomerase I (Ab109374; 1:2000; RRID:AB_10861978), α-β-Actin (Sigma A2228; 1:5000; RRID:AB_476697)) overnight at 4°C followed by washing thrice (10 min each) in 0.1% Tween/TBS. Membranes were incubated with appropriate HRP-conjugated secondary antibodies (Polyclonal goat anti-mouse (Dako, P0447; RRID:AB_2617137) and polyclonal goat anti-rabbit (Dako, P0448; RRID:AB_2617138)) at room temperature for 1 h and washed thrice (10min each) prior to signal detection. EMD Millipore Immobilon Western Chemiluminescent HRP Substrate (ECL) was used for detection.
4
Protein Extraction and Quantification
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Total protein was extracted using Pierce™ IP Lysis Buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) (Thermo Fisher Scientific, Waltham, MA, USA) with freshly added Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturers’ requirement. Prior to our analyses, total protein concentration was measured using a Bradford reagent (Protein assay dye concentrate, Bio-Rad Laboratories; Hercules, CA, USA) and calculated against a standard curve of standard bovine serum albumin (BSA) (Thermo Fisher Scientific, Waltham, MA, USA) dilutions.
5
Protein Quantitation and Western Blot
6
Western Blot Analysis of CD3ζ
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Cell lysates were prepared by re-suspending the cells in RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0) containing Protease Inhibitor Cocktail (Roche), followed by incubation on ice for 20 mins. The lysates were sonicated on ice (5 W, 10 × 3 s), and insoluble components were removed by centrifugation at 12 000 × g for 15 min. The supernatant was collected and protein concentrations were determined with Bio-Rad Protein Assay Dye Concentrate. Proteins were separated by SDS PAGE gels (10–15% acrylamide) and then transferred onto PVDF membranes with 0.2 μm pore size (Bio-Rad). Nonspecific binding was blocked by 4% skim milk in 1× PBST (1× PBS, 0.05% Tween20) for 1 h at room temperature and then incubated overnight at 4°C with mouse anti-human CD3ζ primary antibody (1:1000 dilution; clone 8D3; Pharmingen) in 1× PBST containing 5% BSA. Amounts of ß-actin protein were determined by monoclonal antibodies raised against human ß-actin (1:10 000 dilution; A1978, Sigma). Blots were washed with 1× PBST and incubated for 2 h at room temperature with goat anti-mouse IgG HRP conjugate secondary antibody (1:3000, R&D systems) in 4% skim milk. After washing with 1× PBST, immunoreactive bands were detected using the Western HRP substrate (Luminata Forte, Millipore) in an infrared Imager (LAS-4000, Fuji).
7
Western Blot Protocol for Protein Expression
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Total protein extracts were obtained by homogenization in 50 mM Tris/HCl pH 8.0, 150 mM NaCl, 0,1% SDS, 0,5% sodium deoxycholate, 1% NP-40 adjusted with protease and phosphatase inhibitors. The homogenate was centrifuged at 13,400 rpm at 4 °C for 10 min. The supernatant was collected and protein content quantified using Protein assay dye concentrate (BIO-RAD). Thirty μg of protein from total extracts, were adjusted with Laemmli Buffer (Sigma) and loaded on acrylamide/bis-acrylamide gels. Gel electrophoresis, transfer to PVDF membrane (Millipore) and signal development with chemiluminescence substrate ECL for HRP (PerkinElmer) were performed as previously described [60 (link)]. Primary antibodies were used at the following dilutions: CDC6 (Santa Cruz #9964) 1:1000, p-RB (Santa Cruz #7986-R) 1:500, RB (Santa Cruz #50) 1:1000, p53 (Santa Cruz #47698) 1:1000, p21WAF1/Cip1 (Santa Cruz #6246, 1:400), Actin (Cell Signaling #4967, 1:1000). Anti-mouse (Cell Signaling #7076) and anti-rabbit (Cell Signaling #7074) HRP-linked secondary antibodies diluted at 1:1000 were used.
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