Wild type or AAAA versions of the RHIM-containing regions of human RIPK3 (Q9Y572; residues 387–518), human ZBP1 (Q9H171; residues 170–355) and VZV (strain Dumas) ORF20 (P09276; residues 1–114) were expressed as His-tagged N- or C-terminal YPet, mCherry or ubiquitin fusion proteins (S6 Fig ) in E. coli BL21(DE3) pLysS (Novagen) grown in LB media containing ampicillin. Protein expression was induced with 0.5 mM Isopropyl β-D-thiogalactoside (IPTG) when the OD600 nm of the culture reached 0.6–0.8 for 3 h at 37 oC. All fusion proteins were purified from inclusion bodies under denaturing conditions using Ni-NTA agarose beads (Life Technologies) [39 (link)]. Purified proteins were concentrated with Amicon Ultra-15 MWCO 30,000 centrifugal filter units and stored in 8 M urea, 20 mM Tris, pH 8.0. Protein concentration was determined using the Pierce Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific).
Pierce bicinchoninic acid protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany, France
The Pierce bicinchoninic acid (BCA) protein assay kit is a colorimetric detection and quantitation method for total protein. It relies on the reduction of Cu2+ to Cu+ by protein in an alkaline medium, with the detection of the Cu+ by the reagent bicinchoninic acid.
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Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Chemidoc xrs system, by Bio-Rad (4 mentions)
Amersham imager 600, by GE Healthcare (3 mentions)
Amersham imager, by Cytiva (3 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor, by Roche (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (3 mentions)
Cell lysis buffer, by Cell Signaling Technology (3 mentions)
Ripa buffer, by Merck Group (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Ripa buffer, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Santa Cruz Biotechnology (2 mentions)
Fluorchem fc2 imaging system, by Bio-Techne (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence western blotting detection reagent, by GE Healthcare (2 mentions)
Anti gapdh, by Abcam (2 mentions)
118 protocols using pierce bicinchoninic acid protein assay kit
1
Purification of RHIM-containing Proteins
2
Western Blot Protocol for Protein Analysis
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First, cells were homogenized in radio‐immunoprecipitation assay lysis buffer (R0010; Solarbio) containing 1% phenylmethanesulfonyl fluoride on ice for 30 minutes. Total protein concentrations were estimated using the Pierce bicinchoninic acid protein assay kit (23225; Thermo). Then, equal amounts of cell lysates were loaded and separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Then, proteins were transferred to nitrocellulose membranes for 1 hour with a 300 mA current. Next, the membranes were incubated with a 1:1000 dilution of anti‐HNRNPK antibody (ab52600; Abcam) and a 1:10 000 dilution of anti‐β‐actin antibody (ab49900; Abcam) overnight at 4°C with 5% nonfat milk, washed three times with tris‐buffered saline tween 20 buffer, and then incubated with a 1:10 000 dilution of horseradish peroxidase‐conjugated goat anti‐rabbit Immunoglobulin G (ZDR‐5307; ZSGB‐Bio) for 1 hour at room temperature. The membranes were visualized with an emitter coupled logic detection system (Thermo).
3
Western Blot Analysis of PTEN and PI3K Signaling
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Cells were lysed with Lysis Buffer (Beijing Solarbio Science & Tecnology Co., Ltd., Beijing, China) and total protein concentration was measured using the Pierce Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of protein (50 µg) were separated by 8% SDS-PAGE and transferred to polyvinylidene fluoride membranes, which were blocked with ice-cold transfer film buffer (0.29% Tris-base, 0.58% glycine, 0.037% SDS and ddH2O) for 60–90 min. Primary antibodies against PTEN (1:1,000), p-PI3K (1:1,000), PI3K (1:1,000), p-AKT (1:1,000), AKT (1:1,000), ABCG2 (1:1,000) and β-actin (1:1,000) were applied overnight at 4°C at a dilution of 1:1,000. Proteins were detected by enhanced chemiluminescence (EMD Millipore, Billerica, MA, USA), following incubation with horseradish peroxidase-conjugated secondary antibodies (1:10,000 dilution; cat. no. HAF008; R&D Systems China Co., Ltd., Shanghai, China) at room temperature for 1 h. Blots were semi-quantified using ImageJ (V1.8.0; National Institutes of Health, Bethesda, MD, USA).
4
Quantitative Western Blot Analysis of Cardiac Proteins
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Total proteins were extracted from the cardiac tissues or cardiac fibroblasts using radioimmunoprecipitation assay lysis buffer (150 mM NaCl, 1% Triton X‐100, 1% sodium deoxycholate, 50 mM Tris–HCl [pH 7.5], 2 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 mM Na3VO4 and 5 mM NaF) supplemented with a protease inhibitor cocktail (Calbiochem/EMD Millipore). The protein concentration was determined using the Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Equal amounts (40 μg) of proteins were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis using a 10% gel. The resolved proteins were transferred onto a polyvinylidene difluoride membrane (pore size = 0.45 μm), and the membrane was blocked with 5% skim milk and incubated with the primary antibodies (1:1000) at 4°C overnight. Next, the membrane was washed three times with Tris‐buffered saline containing Tween 20 buffer (20 mM Tris, 200 mM NaCl and 0.04% Tween 20) to remove unbound proteins. It was then incubated with anti‐rabbit or anti‐mouse horseradish peroxidase–conjugated secondary antibodies (1:5000) for 1 h at 25°C. Immunoreactive signals were visualized using Immobilon Western blotting detection reagents (EMD Millipore), and the protein band intensities were quantified using ImageJ (National Institutes of Health).
5
Pharmacological Modulation of Cell Signaling
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MHCC97-H cells (5x106 cells per 75T flask) were incubated for 24 h following pretreatment with solvent control, compound 4 , LY294002, GSK2118436, CP690550 and rapamycin for 12 h. The dosing concentrations of solvent control, compound 4 , LY294002, GSK2118436, CP690550 and rapamycin all were set to 1 μmol/L. At the end of the exposure period, cells were lysed in RIPA (Radio Immunoprecipitation Assay) buffer (Cell Signaling Technology, Danvers, MA, USA). Protein concentration was determined using the Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. Protein samples (40 μg) were loaded into each well of a 10–12% polyacrylamide gel and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Millipore, Billerica, USA). The membrane was blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 and incubated with antibodies (1:1000) at 4°C. β-actin was employed as the loading control. Rabbit antibodies against human phospho-p70S6K (Thr389), p70S6K, phospho-AKT (Ser473), phospho-AKT (Thr308), AKT, phospho-ERK, ERK, phospho-rpS6 (Ser235/6), rpS6, β-actin and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).53 (link)–55 (link)
6
Western Blot Analysis of IGF1R Protein
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Whole-cell lysates were prepared using RIPA buffer (Cell Signaling Technology, Inc.). The protein determination used Pierce Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of total protein (30 µg) were separated by 10% SDS/PAGE, transferred to a PVDF membrane (EMD Millipore) and detected using an ECL Western Blotting Detection System (Bio-Rad Laboratories, Inc.). 5% skim milk was used to block the membrane at room temperature for 1 h. Primary rabbit polyclonal antibodies against IGF1R (ProteinTech Group, Inc.; cat. no. 10297-1-AP; dilution, 1:200) were used at 4°C overnight. An antibody against GAPDH (ProteinTech Group, Inc.; cat. no. 60004-1-Ig; dilution, 1:5,000) was used as the loading control. Goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP (OriGene Technologies, Inc.; cat. nos. TA130024 and TA130005; dilution, 1:5,000) secondary antibodies were used at room temperature for 1 h (20 (link)). The software used for densitometry was ImageJ version 1.46 (National Institutes of Health).
7
Protein Expression Analysis by Western Blot
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Total protein was extracted from the tissues and cells using a RIPA buffer (Thermo Fisher Scientific, Inc.). The protein concentrations were determined with a Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). The proteins were separated using 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Thermo Fisher Scientific, Inc.). After blocking with 5% skimmed milk overnight at 4°C, the membranes were incubated with the primary antibodies against caspase-3 (cat. no. ab13847; 1:500), Bcl2 (cat. no. ab32124; 1:200), Bax (cat. no. ab32503; 1:500), matrix metalloproteinase [(MMP)2; cat. no. ab97779; 1:200], MMP9 (cat. no. ab38898; 1:500), N-cadherin (cat. no. ab76057; 1:500), vimentin (cat. no. ab45939; 1:500), E-cadherin (cat. no. ab227639; 1:200), and GAPDH (cat. no. ab9485; 1:500) at room temperature for 5 h and then with the HRP-conjugated goat anti-rabbit secondary antibody (cat. no. ab6721; 1:10,000) at room temperature for 2 h (all antibodies were obtained from Abcam). A western lightning™ chemiluminescence reagent plus kit (Thermo Fisher Scientific, Inc.) was used to visualize the bands, while the ImageJ software v1.48 (National Institutes of Health) was used to determine the protein expression level.
8
Western Blot Analysis of IGFBP5 Expression
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Cell lysates were prepared by extracting proteins with RIPA (Beyotime, Shanghai, China). Protein concentration was quantified using the Pierce Bicinchoninic Acid Protein Assay Kit (Thermo Scientific). Equal amounts of protein (30 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoide (PVDF) membranes (Millipore, Bedford, MA, USA). After blocked in 5% skim milk for 2 hours, the membranes were incubated with mouse-anti human IGFBP5 polyclonal antibody (1:500 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and mouse-anti human GAPDH polyclonal antibody (1:2,000 dilution; Santa Cruz Biotechnology Inc.) overnight at 4°C. Then membranes were incubated with the goat anti-mouse IgG linked to a horse radish peroxidase (Santa Cruz Biotechnology Inc.) for 2 hours at 37°C. Protein bands were detected using enhanced chemiluminescence (ECL) reagent (Amersham, GE Healthcare, Velizy-Villacoublay, France) and exposed on an X-ray film. Endogenous control was GAPDH. Gray analysis by image analysis was performed using the software Gel-Pro Analyzer (United States Biochemical, Cleveland, OH, USA) after scanning. The semi-quantitative analysis was performed according to the relative expression of objective protein and internal control protein GAPDH, namely the ratio of objective protein and GAPDH.
9
Western Blot Analysis of Protein Expression
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Cells or tissues were incubated on ice with lysis buffer [50 mM Tris-HCl (pH 7.5), 20 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 5% glycerol, and protease inhibitors] and centrifuged at 20,000 × g at 4°C for 15 min. Supernatants were collected, and their protein concentrations were determined with a Pierce Bicinchoninic Acid Protein Assay Kit (Thermo Fisher, Waltham, MA, USA). Equal amounts of protein (30 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking for 1 h with 5% skimmed milk in Tris-buffered saline (TBS; 10 mM Tris and 150 mM NaCl), the membranes were incubated with the following primary antibodies overnight at 4°C, all of which were raised in mice and supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA): anti-CREB1 (1:1,000 dilution), anti-B-cell lymphoma 2 (BCL-2; 1:800), anti-matrix metalloproteinase9 (MMP9; 1:1,000), and anti-GAPDH (1:2,000). The membranes were subsequently washed three times with TBS and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:5,000; Santa Cruz Biotechnology) for 2 h at room temperature. Immunoreactive protein bands were detected with an enhanced chemiluminescence-based FluorChem® FC2 imaging system (Alpha Innotech, San Jose, CA, USA).
10
Protein Quantification and Immunodetection
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Protein quantification was performed using a Pierce bicinchoninic acid protein assay kit (ThermoFisher), with electrophoresis using SDS-PAGE and immunodetection using calnexin (Abcam, ab22595) and ATP5A (Abcam, ab14748) followed by fluorescent DyLight secondary antibodies (Invitrogen, SA5-10036 and 35519) and visualisation using a CLx imaging system scanner (LI-COR Bioscience), using Geneflow BLUeye protein ladder (245kDa) ladder (Supplementary Fig. 1 ).
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