Total proteins, nuclear and cytoplasmic proteins were extracted from liver tissues as described previously22 (link). In short, total protein and nuclear/cytoplasmic protein extracts of liver tissues were respectively homogenized with RIPA lysis buffer containing 1% 100 mmol/L PMSF (Cat# R0127, Biocolors, Shanghai, China) and NE-PER Nuclear and Cytoplasmic Extraction Reagents (Cat# 78835, Thermo Fisher Scientific). Protein concentration was determined using Pierce BCA Protein Assay Reagent Kit (Cat# 23225, Thermo Fisher Scientific). Proteins (30 μg/lane) were separated by 10% SDS-PAGE and transferred to NC membranes (Cat# 66485, Pall, New York, USA). The membranes were blocked using 5% skim milk (Cat# 1172GR500, BioFroxx, Einhausen, Germany) in TBST [Tris-buffered saline (TBS) containing 0.1% Tween-20] for 1 h at room temperature, and then incubated with different antibodies at 4 °C overnight. The blots were incubated by anti-rabbit IgG, HRP-linked antibody or anti-mouse IgG, HRP-linked antibody for 1 h at room temperature after washing with TBST three times the next day. Immunoreactive bands were visualized by chemiluminescence (Cat# WBKLS0500, Millipore, Burlington, VT, USA) following three washes with TBST.
Pierce bca protein assay reagent kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce BCA Protein Assay Reagent Kit is a colorimetric detection and quantitation assay for measuring total protein concentration. The kit utilizes the bicinchoninic acid (BCA) method to detect and quantify the total protein content in a sample.
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Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Western blot detection system cat no w1008, by Promega (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Skim milk, by neoFroxx (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
Qiashredder column, by Qiagen (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Chemiluminescence detector, by Bio-Rad (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Abcam (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Hybond nitrocellulose membrane, by GE Healthcare (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
P mek1 2, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Col3a1, by Abcam (1 mentions)
Anti col1a1, by Abcam (1 mentions)
17 protocols using pierce bca protein assay reagent kit
1
Extraction and Western Blot Analysis of Liver Proteins
2
Isolation and Fractionation of Kidney Organelles
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The kidney was divided into three regions, the cortex, outer medulla and inner medulla, under a stereoscopic microscope. Each region of kidney was homogenized in an ice-cold isolation solution (300 mM sucrose, 1.3 mM EDTA, 25 mM imidazole, complete protease inhibitor cocktail tablet) for 10 min using a shaker-type homogenizer at 1500 rpm for 10 min (Shakemaster Neo, Bio Medical Science, Tokyo, Japan). The homogenate was centrifuged at 1000× g for 10 min at 4 °C, and the supernatant was subsequently ultra-centrifuged at 200,000× g for 1 h. The pellet obtained from ultra-centrifugation was suspended in the isolation solution, and this suspension was mixed with 4 × sample buffer. This mixture was thereafter incubated at 37 °C for 30 min. The protein concentration in a small amount of suspension solution from each pellet before addition of the sample buffer was determined using the Pierce BCA Protein Assay reagent Kit (Thermo Fisher Scientific Inc., Rockford, IL, USA).
3
Semiquantitative Immunoblotting of AQP2 and pAQP2
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The cell lysates were obtained in 1X Laemmli buffer (10 mM Tris-HCl, pH 6.8, 1.5% SDS), including protease and phosphatase inhibitor cocktail (HaltTM Protease and Phosphatase Inhibitor Cocktail 100X, 78440, Thermo Fisher Scientific, Waltham, MA, USA). They were placed in the QIAshredder column (79656, QIAGEN, Hilden, Germany) and centrifuged at 10,000× g for 2 min at room temperature. The total protein concentration was measured using the BCA assay kit (Pierce BCA protein assay reagent kit; 23227, Thermo Fisher Scientific, Waltham, MA, USA). Semiquantitative immunoblotting was performed, as previously described [21 (link),22 (link)]. Primary antibodies used were anti-AQP2 (1:1000, AB3274, Merk Millipore, Burlington, MA, USA), anti-phosphorylated-AQP2 at serine 256 (1:1000, K0307AP) [30 (link)], and anti-β-actin (1:400,000, A1978, Sigma, St. Louis, MO, USA). Immunoblots were visualized by horseradish peroxidase-conjugated secondary antibodies (P447, P448, DAKO, Glostrup, Denmark). Densitometric values were corrected by the densitometry value of β-actin and band density was quantitated by ImageJ (NIH).
4
Western Blot Analysis of Cardiac Proteins
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The mice cardiac tissues (60-80 mg) or rat cardiac myoblast H9c2 cells were lysed in RIPA buffer (Thermo Fisher Scientific, Inc.). The supernatant was quantified with Pierce BCA protein assay reagent kit (Thermo Fisher Scientific, Inc.). Total protein (20 µg per sample) was loaded and separated in 10% or 12% SDS-PAGE, and transferred to a PVDF membrane (MilliporeSigma). After blocking in TBST (0.1% Tween-20) containing 5% fat-free milk at room temperature for 1 h, the membrane was incubated with primary anti-RUNX1 (1:1,000; cat. no. ab229482; Abcam), anti-DRD2 (1:500; cat. no. bs-20730R; BIOSS), anti-phosphorylated (p-)P65 subunit of NF-κB (1:1,000; cat. no. ab76302; Abcam), anti-NF-κB P65 subunit (1:1,000; cat. no. bs-20355R; BIOSS), anti-TNF-α (1:500; cat. no. bs-2081R; BIOSS), anti-IL-6 (1:500; cat. no. ab259341; Abcam) or anti-β-tubulin (1:1,000; cat. no. ab18207; Abcam) antibodies respectively, overnight at 4˚C. After washing three times in TBST, the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:10,000; cat. no. ab191866; Abcam) for 1.5 h at room temperature. Protein band signals were detected using ECL program (Thermo Fisher Scientific, Inc.) under chemiluminescence detector (Bio-Rad Laboratories). The expression of target protein was normalized to β-tubulin using Image Lab Software (Bio-Rad Laboratories).
5
Evaluation of Vaginal Proteolytic Activity
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Supernatants of vaginal lavage fluids from C3H and CD-1 mice collected at 0, 24, 48, and 96 h post-inoculation were evaluated for proteolytic activity using a Pierce Fluorescent Protease activity assay kit (Thermo) designed for detecting the level of digested FITC-labeled casein as a measurable substrate. Resultant values were normalized to the protein content (per milligram) of each sample measured by the Pierce BCA protein assay reagent kit (Thermo). The proteolytic activity of trypsin (1 µg) was measured in parallel and used as a positive control. Results were expressed as RFI/mg protein ± SEM.
6
Protein Quantification and Western Blot Analysis
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The cells were lysed with RIPA buffer, and the Pierce BCA Protein Assay Reagent Kit (cat No. 23225, Thermo Fisher, Waltham, MA) was used to measure the protein concentration. Approximately 10 μg of protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresisand transferred to a PVDF membrane (Millipore, Bedford, MA). S1 Table summarizes the antibodies used for protein detection. Equal loading of the protein samples was verified with an antibody to β-actin. Immunoreactive signals were detected with the Promega Western Blot Detection System (cat No. W1008, Promega, Madison, WI).
7
Quantification of Growth Factors in BMSCs
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The bFGF, TGF-b, VEGF, and PDGF concentrations were measured using an enzyme-linked immunosorbent assay (ELISA). For this assay, the treated BMSCs were collected and washed with ice-cold PBS. After 48 h, culture supernatants were divided into 200-μL samples in triplicate. Finally, these samples were homogenized in pre-chilled lysis buffer (20 mM HEPES, 0.5 mM EGTA, 1 mM DTT, and 0.32 M sucrose; PH 7.4) containing protease inhibitors cocktail and then incubated at 4 °C for 30 minutes. The samples were centrifuged at 15, 000 rpm in a pre-chilled centrifuge at 4 °C for ten minutes. The supernatant from the cell lysate was collected and assayed for protein concentration using the Pierce™ BCA Protein Assay reagent kit (Thermo Fisher Scientific, Waltham, MA, USA). Samples containing equal amounts of total proteins were analyzed by ELISA kits for bFGF, TGF-β, VEGF, and PDGF according to the manufacturers’ protocols. The sensitivity of these kits was less than 1 ng/mL, the intra-assay coefficient of variation was 10%, and the inter-assay coefficient of variation was 10%. All the reported results are based on three independent experiments on separate batches of cells.
8
Western Blot Analysis of Signaling Proteins
9
Inflammatory Cytokine Profiling in Spinal Cord Injury
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Proteins were extracted from spinal cord segments (10-mm length) containing the injury site using ultrasonication in all groups, on days 3, 7, and 14 after surgery (5 rats/group). The protein extract was obtained using centrifugation for 20 minutes at 12,000 g and 4°C. The concentration of inflammatory cytokines in the supernatant was then measured using enzyme-linked immunosorbent assay (ELISA) kits (TNF-α, IL-1β, IL-6, and IL-10; Abcam, London, UK) in accordance with the manufacturer’s instructions. The concentration of each protein in each sample was normalized based on the total protein concentration determined using the bicinchoninic acid method (Pierce BCA protein assay reagent kit, Thermo Fisher Scientific Inc.).
10
Protein Expression Analysis Protocol
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Total proteins were extracted from tissues and cells after 72 h cultivation by using lysis buffer (20 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA) and quantified by Pierce BCA Protein Assay Reagent Kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.) according to the manufacturer’s protocol. Equal amount of protein was separated on denaturing SDS gel and transferred to a PVDF membrane. The membrane was blocked with 10% skim milk and the incubated with primary antibodies overnight at 4°C, followed by incubation in appropriate horseradish peroxidase (HRP)–conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.). The membrane was washed twice with PBS and blots were then visualized by ECL system (Bio–Rad Laboratories, Hercules, CA, U.S.A.). GAPDH was used as loading control. The specific primary antibodies were as follows: anti-COL1A1, COL3A1, Timp-1, MMP-3, Cyclin D1, c-fos (Abcam, U.S.A.), MEK1/2 and p-MEK1/2, ERK1/2 and p-ERK1/2 (Cell Signaling, Beverly, MA, U.S.A.).
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