Cells were lysed, resolved by SDS-PAGE, and transferred to nitrocellulose filter membranes (Biorad, Milan) [27 (link)]. After blocking, membranes were incubated with: anti-PD-L1 (R&D Systems, Milan, Italy), -pSTAT3, -STAT3, -pERK1/2 and -ERK1/2 (all from Cell Signaling Technology, Danvers, MA). After incubation with horseradish peroxidase-conjugated secondary antibody (PerkinElmer, Milan, Italy), reaction was visualized with ECL using ImageQuant LAS4000 (GE Healthcare, Milan, Italy).
Nitrocellulose filter membrane
Manufactured by Bio-Rad
Sourced in United States
Nitrocellulose filter membranes are a type of laboratory filtration material used for various applications. They are made from a thin, porous sheet of nitrocellulose material and are designed to efficiently separate and retain specific molecules or particles from a solution or suspension. The core function of these membranes is to facilitate filtration and separation processes in a variety of laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (2 mentions)
γ tubulin, by Merck Group (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by PerkinElmer (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Anti pd l1, by R&D Systems (1 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
Ta 09, by ZSGB-BIO (1 mentions)
Image lab 5, by Bio-Rad (1 mentions)
Ab125066, by Abcam (1 mentions)
Ab137550, by Abcam (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by ZSGB-BIO (1 mentions)
X ray film, by Fujifilm (1 mentions)
Ecl system, by Cytiva (1 mentions)
17 protocols using nitrocellulose filter membrane
1
Western Blot Analysis of PD-L1, STAT3, and ERK
2
Western Blot Analysis of Oocyte and Cumulus Cell Proteins
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According to previous study (Liu et al., 2015 (link)), total protein was extracted from 200 denuded oocytes and the corresponding cumulus cells immediately after collection. For protein extraction, samples were treated in 2 × Laemmli sample buffer and boiled for 10 min followed by cooling on ice. Total proteins were separated by SDS-PAGE and transferred to nitrocellulose filter membranes (0.45-μm pore size, Bio-Rad Laboratories, Richmond, CA, United States). The membrane was blocked in Tris-buffered saline Tween-20 (TBST, TBS with 0.05% Tween 20) containing 5% (w/v) non-fat dry milk for 2 h, and then incubated with the anti-CASR (1:300 dilution, sc32181, Santa Cruz Biotechnology, Santa Cruz, CA) or anti-β actin (1:1,000 dilution, TA-09, ZSGB, Beijing, China) primary antibodies in TBST containing 5% (w/v) non-fat dry milk for 2 h at room temperature. After three 10-min washes in TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated donkey anti-goat IgG (1:10,000) and goat anti-rabbit IgG (1:2,000) secondary antibodies for CASR and ACTIN, respectively, for 1 h at room temperature. Immunoreactive signals were detected with an enhanced chemiluminescence kit (Merck Chemical Co., Darmstadt, Germany) according to the manufacturer’s instructions.
3
Western Blot Analysis of Lysine Modifications
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Protein lysate (20 μg) generated from the tachyzoites of both the RH and ME49 T. gondii strains was separated using 12% Bis-Tris polyacrylamide gels and then transferred to nitrocellulose filter membranes (Bio-Rad, Hercules, CA). Next, 5% skim milk using Tris-buffered saline solution with Tween (TBST) was used to block the membrane for 1 hour at 37 °C, and then the membrane was incubated with antibodies to crotonyllysine (Cat#: PTM-502) (1: 2000; PTM BIO) and 2-hydroxybutyryllysine (Cat#: PTM-801) (1: 1000; PTM BIO) at 4 °C overnight. The membrane was washed three times using TBST buffer before incubation with a secondary antibody at 37 °C for 1 hour (1:10,000; Thermo Scientific™ Pierce, 31430, 31460, MA).
4
Western Blot Analysis of Gpx4 and Nrf2
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Western blot analysis was used as formerly reported (Jiang et al., 2014a). Briefly, aliquots of protein were transferred onto nitrocellulose filter membranes (Bio-Rad, Hercules, CA, USA) after SDS-PAGE electrophoresis. After blocking with nonfat milk for 1 hour, nitrocellulose filter membranes were incubated with primary antibodies (rabbit anti-mouse Gpx4 antibody, 1:1000, ab125066, Abcam, Shanghai, China; rabbit anti-Nrf2 antibody, 1:1000, ab137550, Abcam) overnight at 4°C. Peroxidase-conjugated goat anti-rabbit IgG (ZSGB-BIO, Beijing, China) was diluted 1:500 and incubated at 25°C for 1.5 hours. Protein bands were analyzed by Image lab 5.2.1 (Bio-Rad).
5
Western Blot Analysis of Protein Expression
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Cells were lysed using RIPA buffer [50 mM Tris–HCl (pH 7.4), 1.0 % NP-40, 0.25 % Na-deoxycholate, 1 mM EDTA, 150 mM NaCl, 1 mM aprotinin, 1 mg/ml PMSF, 1 µg/ml pepstatin and 1 µg/ml leupeptin]. The total protein concentrations in the cellular extracts were measured using the BCA assay kit from Keygen Biotech. Co., Ltd. (Nanjing, China). After separation via 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis, the proteins were transferred to nitrocellulose filter membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked using 5 % BSA in Tris-buffered saline containing Tween 20 at 4 °C overnight. The membranes were probed using various primary antibodies at 4 °C overnight, followed by incubation in horseradish peroxidase-conjugated secondary antibodies at 37 °C for 1.5 h. The membranes were exposed to an ECL system (Amersham Biosciences, Uppsala, Sweden), and chemiluminescence was detected by exposing the membranes to x-ray film (Fujifilm Co., Ltd., Tokyo, Japan). The results were digitized using Image Quant 5.2 software (Amersham), and the gray values of the bands were semi-quantitatively evaluated using Gel-Pro Analyzer 4.0 software (Media Cybernetics, Rockville, MD, USA). The gray values were normalized to those of GAPDH.
6
Signaling Pathways in Breast Cancer
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Trastuzumab (Herceptin®) was obtained from F. Hoffmann-La Roche Ltd.; Antibodies for western blot against EGFR, p-EGFR (Tyr1068), HER2, p-HER2 (Tyr1248), HER3, p-HER3 (Tyr1289), Akt, p-Akt (Ser473), ERK1/2, p-ERK1/2 (Thr202/Tyr204), Src, p-Src (Tyr416), IGF-1R, p-IGF-1R (Tyr1135/Tyr1136), GAPDH and corresponding secondary antibodies were purchased from Cell Signaling Technology; PE conjugated anti-EGFR and anti-HER3, FITC-conjugated Annexin V antibodies and propidium iodide (PI) were from eBioscience; PE conjugatedanti-HER2 antibody was from BD; Electrophoresis reagents and Hybridization Nitrocellulose Filter membranes were from Bio-Rad; BCA protein assay and enhanced chemiluminescent (ECL) reagents were from Pierce; Cell culture medium Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone; Human IGF-1, NRG1-β1/HRG1-β1 was from R & D; IGF-1R expressing plasmid pCMV6-IGF1R was from OriGene; Lentiviral delivery system was packaged by Gene Pharma (China); MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and agarose (cell culture level) were purchased from Sigma-Aldrich; Matrigel and Transwell chamber was from Millipore. All other chemicals were obtained from commercial source of analytical grade.
7
HCoV-OC43 Infection and Protein Expression Analysis
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Cells (1 × 105 cells/24-well plate; Thermo Fisher Scientific Inc.) were infected with HCoV-OC43 and treated with the indicated compounds until 4 dpi at 33 °C. Cells lysates were then separated on SDS–PAGE gel and transferred to nitrocellulose filter membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA), which were incubated with 5% skim milk in TBST (0.5% Tween20) for 30 min at room temperature and then rinsed three times with cold TBST. The anti-HCoV-OC43 nucleocapsid protein antibody (Catalogue Number. MAB9012, Merck & Co., Inc., Rahway, NJ, USA), anti-HCoV-OC43 spike protein antibody (Cat. No. CSB-PA336163EA01HIY, Cusabio, Houston, TX, USA), anti-phospho-p38 MAPK antibody (Cat. No. 9211S, Cell Signaling Technology, Danvers, MA, USA), anti-total p38 MAPK antibody (Cat. No. 9212S, Cell Signaling Technology), or anti-β-actin antibody (Cat. No. 3700S, Cell Signaling Technology) was then added and incubated at 4 °C overnight. After washing, horseradish peroxidase-conjugated secondary antibodies (Abcam PLC, Cambridge, UK) were added and incubated for 2 h at room temperature. Proteins were detected using a ChemiDoc™ MP Imaging System (Bio-Rad Laboratories). Band intensity was measured using the ImageLab program (Bio-Rad).
8
Western Blot Immunodetection of Cellular Proteins
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Whole cells or subcellular fractions obtained as described above, were resolved by SDS-PAGE, and transferred to nitrocellulose filter membranes (Biorad, Milan, cod. 1704158) (2). After blocking (5% Not-fat dry milk, Santa Cruz Biotechnology, Heidelberg, Germany, cod. sc2325), membranes were incubated with: anti-GFP (Cell Signaling Technologies, Danvers, MA, cod 2555S), anti-vinculin (Abcam, Cambridge, UK, cod. 130007), -tubulin (Cell Signaling Technologies, cod.2114), -actin (Santa Cruz Biotechnology, cod. sc-47778), -Hadha (Abcam, cod. 203114), -cytochrome C (BD Bioscience, cod. 556433), -H2A (Abcam, cod. ab18255), anti-NAMPT (Bethyl Laboratories, Montgomery, TX A300-779A), anti-NAPRT1 (Novus Biologicals, Cambridge, UK, cod. NBP1-87243), anti-C9orf95 (NRK1, ab169548) and anti-QPRT (ab57125 both from Abcam). After incubation with horseradish
9
Quantification and Western Blot Analysis
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Total protein was extracted and quantified using a BCA Protein Assay Kit (Pierce Biotechnology Inc., Rockford, IL, USA). Equal amounts of protein were separated by SDS-PAGE (15% for Tβ4 detection), then transferred onto nitrocellulose filter membranes (Bio-Rad, Hercules, CA, USA) and blocked with 5% nonfat milk. The following antibodies were used: anti-Tβ4 (1:1000, Santa Cruz Biotechnology), anti-NF-κB p65 (1:1000, Santa Cruz Biotechnology), anti-p-ERK (1:2000, Cell signaling technologies), anti-ERK (1:2000, Cell signaling technologies), anti-p-p38 (1:500, Cell signaling technologies), anti-p38 (1:1000, Cell signaling technologies), anti-p-JNK (1:1500, Cell signaling technologies), anti-JNK(1:1000, Cell signaling technologies). Horseradish peroxidase-conjugated were used as secondary antibodies. Target proteins were detected according to standard methods and β-actin was used as an internal control.
10
Western Blot Analysis of Integrin β1, PI3K, and Akt
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Cells were lysed in cold RIPA lysis buffer supplemented with Protease Inhibitors cocktail (CST Inc., USA). The protein concentration was quantified using BCA protein assay kit (Beyotime, China) following the manufacturer’s introduction. Twenty micrograms of protein were mixed with 1× loading buffer and boiled for 5 mins at 100°C and were resolved by 8% or 10% SDS-PAGE and transferred to the nitrocellulose filter membrane (Bio-Rad). Target protein was detected using specific primary antibody, including: anti-Integrin β1 (1:1000; Abcam, Cambridge, UK), anti-PI3K (1:1000; Abcam), anti-Akt (1:1000; Abcam), anti-phospho-PI3K (1:1000; Abcam), anti-phospho-Akt (1:1000; Abcam). All experiments were repeated three independent times and the target protein level was quantified by ImageJ and normalized to internal control.
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