Proteins from HCC tissues and cells were processed according to the standard procedure and quantified using a bicin-choninic acid (BCA) protein assay kit (Cat. #23227, Thermo, USA). We added 10 μg of proteins per well, separated them using the 8% or 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred them to PVDF membranes (Bio-Rad, USA). The PVDF membranes were blocked in 5% DifcoTM Skim Milk (Cat. #232100, BD, USA) for one hour and then incubated overnight at 4°C with primary antibodies against CCT6A (Cat. #049949, 1:200, Human Protein Atlas), cyclin D (Cat. #2978, 1:1000, Cell Signaling Technology, USA), and GAPDH (Cat. #60004-1-Ig, 1:1000, Proteintech, China). The membranes were washed three times with TBST buffer at intervals of 15 mins, and were than incubated with the corresponding second antibodies conjugated with horseradish peroxidase (HRP) (Cat. #7074/#7076, Cell Signaling Technology, USA). We perfomed enhanced chemiluminescence (ECL, Pierce) to visualize the bands and used ImageJ software to carry out the quantitative analysis.
Difcotm skim milk
Manufactured by BD
Sourced in United States
DifcoTM Skim Milk is a powdered milk product used as a general-purpose culture medium in microbiological applications. It provides essential nutrients to support the growth of a variety of microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Amersham imager 600, by GE Healthcare (4 mentions)
Bicinchoninic acid method, by Beyotime (3 mentions)
Protease inhibitor cocktail set 3, by Merck Group (3 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Beyotime (3 mentions)
Piercetm bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (2 mentions)
Tween 20, by Merck Group (2 mentions)
Dylight 800 4xpeg conjugated goat anti rabbit igg h l, by Cell Signaling Technology (2 mentions)
Goat anti mouse igg h l, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by BD (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Gapdh, by Proteintech (1 mentions)
Nitrocellulose blotting membrane, by Cytiva (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Coomassie plustm protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Immobilontm western chemiluminescent hrp substrate, by Merck Group (1 mentions)
13 protocols using difcotm skim milk
1
Western Blot Analysis of Hepatocellular Carcinoma
2
Protein Extraction and Western Blot Analysis
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The total protein was extracted from AMVMs or NMVMs. First, AMVMs or NMVMs were lysed by radioimmunoprecipitation (RIPA) buffer with cocktail protease inhibitor (Beyotime Biotechnology, China) at 4 °C. Then the lysis buffer was clarified by centrifugation at 12,000 rpm for 15 min at 4 °C and the supernatant protein concentration was determined using the bicinchoninic acid method (Beyotime Biotechnology, China). Proteins (20 μg) were size-fractionated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto Immobilon polyvinylidene difluoride membranes. The membranes were blocked for 2 h with 5% DifcoTM Skim Milk (BD Biosciences, USA). Then the primary antibodies was added and incubated at 4 °C overnight, and diluted secondary antibody (Cell Signaling Technology, USA) was added and incubated at room temperature for 1 h. The results were visualized and analyzed by Odyssey Infrared Imaging System (LICOR, USA). All primary and secondary antibodies are shown in Additional file 8 : Table S2.
3
Protein Extraction and Western Blotting
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The cells were incubated with CytoBusterTM Protein Extraction Reagent (Novagen, Darmstadt, Germany), protease inhibitor cocktail (Sigma-Aldrich, MO, USA) and phosphatase inhibitor cocktail 2 (Sigma-Aldrich) for 30 min at 4 °C. The cell extracts were then purified by centrifugation at 12,000 rpm for 15 min at 4 °C, and protein concentrations were determined by Coomassie PlusTM Protein Assay Reagent (Thermo Fisher Scientific). Protein samples (15 μg) were mixed with Tricine sample buffer (Protech Technology, Taipei, Taiwan), heated for 5 min at 95 °C, resolved on a 4–12% Bis/Tris NuPAGE gel (Invitrogen, Carlsbad, CA, USA) and transferred to a Nitrocellulose blotting membrane (Amersham Biosciences, Piscataway, NJ, USA) using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories, Hercules, CA, USA). The membranes was subsequently blocked with 5% DifcoTM Skim Milk (BD Biosciences, Franklin Lakes, NJ, USA) in PBS for 1 h, hybridized with the indicated Ab and then incubated with horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG or goat anti-rabbit IgG (Millipore, Billerica, MA, USA) for 1 h before being incubated with ImmobilonTM Western Chemiluminescent HRP Substrate (Millipore). The immuno-reactive signals were stripped by RestoreTM Western Blot Stripping Buffer (Thermo Fisher Scientific) before the protein of interest was re-probed.
4
Quantitative Western Blot Analysis
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Cells were treated with cold sample lysis buffer (1% Triton X-100, Xpert Protease Inhibitor Cocktail Solution (GenDEPOT, Katy, TX, USA), 5 mM Ethylenediaminetetraacetic acid (EDTA, Thermo Fisher Scientific, Waltham, MA, USA), and 1 mM Phenylmethanesulfonyl fluoride (PMSF, Thermo Fisher Scientific, Waltham, MA, USA)). Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used for determining the protein concentration. An equal amount of protein was loaded for SDS-PAGE and transferred onto PVDF membrane using Wet/Tank Blotting Systems (Bio-Rad Laboratories Inc., Hercules, CA, USA). The membranes were incubated with 5% DifcoTM Skim Milk (BD Biosciences, San Jose, CA, USA) for 30 min at room temperature. The membranes were incubated with cleaved caspase-3 (Asp175) antibody (Cat. 9661S, Cell Signaling Technology, Danvers, MA, USA) and GAPDH antibody (Cat. sc-47724, Santacruz Biotechnology Inc., Dallas, TX, USA) overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). We added ECLTM Select Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) on the membrane for the detection of the signals from HRP. The images of protein bands were obtained by a LAS-3000 imaging system (Fujifilm, Minato, Tokyo, Japan).
5
Western Blot for BDNF and NGF Quantification
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Total proteins were extracted in each group using RIPA buffer (CellNest, Minato, Tokyo, Japan) with Protease Inhibitor Cocktail Set III (1:1000, Millipore, Billerica, MA, USA). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), in accordance with the manufacturer’s protocol. Protein samples were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with 5% DifcoTM skim milk (BD Biosciences, Franklin Lakes, NJ, USA) in 1× Tris-buffered saline (TBS, Bio-Rad, Hercules, CA, USA) with 0.1% Tween 20 (Sigma-Aldrich), and probed using various antibodies. The Western blots were visualized using ECL (Bio-Rad) and exposed to the Amersham Imager 600 (GE Healthcare Life Sciences, Uppsala, Sweden). The equivalence of protein loading was verified by probing for actin. The antibodies used were as follows: rabbit anti-BDNF (1:500, Abcam), rabbit anti-NGF (1:200, R&D Systems), mouse anti-β-actin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), horseradish peroxidase-conjugated anti-rabbit antibody (1:2500, Abcam), and horseradish peroxidase-conjugated anti-mouse antibody (1:2500, Abcam).
6
Protein Extraction and Western Blot Analysis
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Proteins were extracted from frozen colorectal tissues and cultured cells using RIPA solution (Code: P0013B, Beyotime, Shanghai) and a protease inhibitor (Code: A32961, Thermo Fisher Scientific, Shanghai, China), NaF (50 mM), PMSF (1 mM), and Na3VO4 (1 mM). All samples were lysed at 4°C after 30 min. The mixtures were then centrifuged at 12,000 g for 15 min at 4°C, and the supernatant was carefully collected. Next, we used the PierceTM BCA Protein Assay Kit (Code: 23227, Thermo Fisher Scientific, Shanghai, China) to measure the protein expression level by absorbance at 570 nm. Then, 30 μg of each protein sample was separated by 10% SDS‐PAGE and electrophoretically transferred to polyvinylidene fluoride membranes. To block nonspecific protein binding, use 5% DifcoTM Skim Milk (Code: 232100, BD, USA) dissolved in TBST overnight at 4°C. Membranes were incubated with primary antibodies overnight at 4°C. After being washed three times with TBST buffer, HRP‐conjugated IgG or IgM (working dilutions: 1:1000; Code: A200 and A21, Beyotime, China) as secondary antibodies, and incubated at room temperature for 1 hour. Antigen and antibody complexes were detected following a Bio‐Rad ChemiDoc™ Touch. Immunoblots were quantified using ImageJ (Quantity One software, Bio‐Rad, CA, USA). All primary antibodies are listed in Supplementary Data S1 .
7
Protein Extraction and Western Blot Analysis
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Cells were lysed in RIPA buffer (CellNest, Minato, Tokyo, Japan) containing protease inhibitor cocktail set III (1:1000, Millipore, Billerica, MA, USA). The concentration of the extracted protein was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) in accordance with the manufacturer’s protocol. The extracted proteins were separated using SDS-PAGE (20 μg proteins/lane), transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with 5% DifcoTM skim milk (BD Biosciences, San Jose, CA, USA) in a buffer solution consisting of 1× tris-buffered saline (TBS, Bio-Rad) with 0.1% (v/v) Tween 20 (TBST), and probed with IGF-1R (1:1000, Cell signaling) and VEGFR (1:500; Novus). After overnight incubation at 4 °C, membranes were washed and then soaked in secondary solutions containing horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies (1:2500, Abcam, Cambridge, UK). Detection was accomplished using ECL (Bio-Rad) and imaged using an Amersham Imager 600 (GE Healthcare Life Sciences, Piscataway, NJ, USA). Equivalent protein loading was verified by probing with β-actin.
8
Protein Extraction and Western Blot Analysis
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Proteins were extracted from mouse AMVMs with ice‐cold modified radioimmunoprecipitation assaybuffer (P0013C, Beyotime Biotechnology, China) containing protease and phosphatase inhibitors. Lysates were centrifugated at 14 000 rcf for 15 minutes at 4 °C and protein lysates were transferred to a new Eppendorf tube for bovine serum albumen quantification. Protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 7.5% to 15% gradient gels (Bio‐Rad, USA) and transferred to polyvinylidene difluoride (IPVH00010, Millipore, Germany) membranes. The membranes were blocked for 2 hours with 5% DifcoTM Skim Milk (BD10610, BD Biosciences, USA) and incubated overnight at 4 °C with primary antibodies: Pgc1‐α (4A8) (1:1000, sc‐517380, Santa Cruz, USA), Pgc1‐β (E‐9) (1:1000, sc‐373771, Santa Cruz, USA), γ‐H2A.X (1:500, 9718S, CST, USA), histone H3 (1:1000, CY6587, Abways Technology, China), p53 (1:1000, 10442‐1‐AP, Proteintech, USA), DRP1 (D6C7) (1:1000, 8570S, Cell Signaling Technology, USA), and OPA1 (D6U6N) (1:1000, 80471S, Cell Signaling Technology, USA) followed by secondary antibody for 1 hour with a 1:10 000 dilution of IgG Goat Anti‐Mouse HRP (SA00001‐1, Proteintech, USA) or IgG Goat Anti‐Rabbit HRP (SA00001‐2, Proteintech, USA). The results were visualized using an Amersham Imager 600 (General Electric Company, USA).
9
Apo-lactoferrin Preparation and Evaluation
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The specific preparation method of apo-LF is as described in the previous patent A method for preparing lactoferrin with required iron saturation [18 ]. Gibco (Carlsbad, CA, USA) provided Dulbecco’s modified Eagle’s medium (DMEM)/F-12 and fetal bovine serum (FBS). Beijing Solarbio Technology Co. Ltd. (Beijing, China) provided LPS, ELISA kit for mouse TNF-α, mouse IL-6, mouse IL-1β and mouse IFN-γ, primary antibody (PFKFB3, NF-κB, TNF-α and β-actin) and Goat Anti-Rabbit IgG/HRP. Primary antibody (PPAR-γ and IL-1β) was obtained from Bioss (Beijing, China). Protease inhibitor cocktail, penicillin-streptomycin solution, phosphate-buffered saline (PBS), BCA protein assay kit, trypsin-EDTA solution with phenol red and RIPA lysis buffer were provided by Beyotime Biotechnology (Shanghai, China). DifcoTM skim milk was obtained from BD-Pharmingen (San Diego, CA, USA).
10
Western Blot Analysis of Mitophagy Proteins
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Total proteins were extracted in each group using RIPA buffer (CellNest, Minato, Tokyo, Japan) with Protease Inhibitor Cocktail Set III (1:1000, Millipore, Billerica, MA, USA). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), in accordance with the manufacturer’s protocol. Protein samples were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with 5% DifcoTM skim milk (BD Biosciences) in 1X Tris-buffered saline (TBS, Bio-rad) with 0.1% Tween 20 (Sigma), and probed using various antibodies. The Western blots were visualized using ECL (Bio-Rad) and exposed to the Amersham Imager 600 (GE Healthcare Life Sciences, Uppsala, Sweden). The equivalence of protein loading was verified by probing for actin. The antibodies used were as follows: rabbit anti-PTEN-induced putative kinase 1 (PINK1, 1:500, Novus Biologicals, CO, USA); mouse anti-parkin (1:1000, Cell Signaling); mouse anti-β-actin (1:1000, Santa Cruz); horseradish peroxidase-conjugated anti-rabbit or -mouse antibodies (1:2500, Abcam plc).
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