Western blot was performed as previously described [29 (link)]. Briefly, the cells were lysed in radioimmunoprecipitation assay (RIPA) buffer consisting 2% protease inhibitor cocktail (Roche) and 1% phosphatase inhibitor (Roche) to obtain the protein. After that, BCA protein assay kit was used to test the protein concentrations. Thirty-five micrograms of total protein of each sample was subjected to SDS-PAGE. After electrophoresis, proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% nonfat milk and incubated with anti-AKR1C1 (Abcam, Cambridge, UK), anti-PPARγ, anti-RUNX2, anti-PGR, anti-AR, or anti-GAPDH (Cell Signaling Technology, Beverly, MA, USA) in Tris-buffered saline-Tween 20 (TBST) at 4 °C overnight. After that, the membrane was washed with TBST buffer, incubated with goat anti-rabbit IgG or goat anti-rat IgG (Abcam), and then washed with TBST again. At last, an ECL Western blot kit (CWBIO) was used to visualize the bands.
Ecl western blot kit
Manufactured by CWBIO
Sourced in China
The ECL Western Blot Kit is a laboratory equipment used for the detection and visualization of proteins in a Western blot analysis. The kit contains the necessary reagents and materials to perform the enhanced chemiluminescence (ECL) detection method, which utilizes a luminescent reaction to generate a signal proportional to the amount of target protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Goat anti rabbit igg, by Abcam (3 mentions)
Anti runx2, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Phosphatase inhibitor, by Roche (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti pparγ, by Cell Signaling Technology (1 mentions)
Anti ar, by Cell Signaling Technology (1 mentions)
Goat anti rat igg, by Abcam (1 mentions)
Anti p65, by Cell Signaling Technology (1 mentions)
Anti p iκbα, by Cell Signaling Technology (1 mentions)
Anti p p65, by Cell Signaling Technology (1 mentions)
Anti iκbα, by Abcam (1 mentions)
Anti p ikk, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg hrp conjugate, by Bio-Rad (1 mentions)
β actin, by Merck Group (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
18 protocols using ecl western blot kit
1
Western Blot Analysis of Protein Expressions
2
Western Blot Analysis of Signaling Pathways
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First, hBMMSCs were washed with cold PBS softly for three times. After that, the cells were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with 1% phosphatase inhibitor (Roche) and 2% protease inhibitor cocktail (Roche). After collecting and centrifugation of the cell lysate at 13362g at 4 °C for 30 min, supernatants were carefully transferred to new tubes, and the BCA protein assay kit was used to determine the protein concentrations. Thirty-five-microgram total protein of each sample was subjected to 10% SDS-PAGE. Proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) after electrophoresis. The membrane was then blocked with 5% nonfat milk and then incubated with anti-IκBα (Abcam, Cambridge, UK), anti-p-IKK, anti-p-IκBα, anti-p-P65, anti-P65, anti-RUNX2, or anti-GAPDH (Cell Signaling Technology, Beverly, MA, USA) in TBS at 4 °C overnight. After that, the membrane was washed with Tris-buffered saline-Tween 20 (TBST) buffer for 3 times and then incubated with goat anti-rabbit IgG (Abcam). After that, the membrane was washed with TBST buffer again. Lastly, an ECL Western blot kit (CWBIO) was used to visualize the bands.
3
GNPDA2 Protein Expression in Transfected ADMSCs
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Detection of GNPDA2 protein expression from transfected ADMSCs after stimulating differentiation to adipocytes for 10 days was carried out by western blot. The procedures were performed according to standard protocols or the manufacturers' instructions. Membranes were blotted with GNPDA2 polyclonal rabbit antibody (1 : 1000 dilution; 17105-1-AP, Proteintech), followed by goat anti-rabbit IgG HRP conjugate (1 : 5000; Bio-Rad Laboratories). Bands were visualized using the ECL Western Blot Kit (CWBIO, Beijing, China).
4
Western Blotting Protocol for KIF20A
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Western blotting was performed as described previously [23 (link)]. Total proteins were extracted with radioimmunoprecipitation assay (RIPA) buffer supplemented with 1% phenylmethanesulfonyl fluoride (PMSF). The protein concentrations were quantified using a BCA Protein Assay Kit (Solarbio, Beijing, China). Equal concentrations were separated by 10% SDS-PAGE and electrotransferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in TBS-T for 1 h at room temperature. Then, the blots were incubated with primary antibodies against human KIF20A (1 : 500, Proteintech, Wuhan, China) and β-actin (1 : 3000, Sigma-Aldrich, USA) overnight at 4°C and then with rabbit or mouse secondary antibodies for 1 hour at room temperature after being washed with TBS-T. After washing, the ECL Western Blot Kit (CWBIO, Beijing, China) was used to detect the specific bands.
5
Epithelial-Mesenchymal Transition Assay
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GK was purchased from MCE co, Ltd. (Shanghai, China). Cell Signaling Technology provided the primary antibodies used in this study: anti-N-cadherin (#13116), anti-Vimentin (#5741), anti-Snail (#3879), anti-p-AKT (#4060), anti-AKT (#4691), anti-GSK-3β (#12456),anti-p-GSK-3β(#5558),anti-actin (#4970), and anti-rabbit IgG HRP (#7074) (Danvers, MA, United States). Anti-c-Myc (ab32072) purchased from Abcam (Cambridge, United Kingdom)). Gibco provided the trypsin-EDTA used in this study (California, United States). CWBio supplied the RIPA Lysis Buffer and the ECL Western blot kit (Taizhou, Jiangsu, China). Hyclone was consulted to acquire the minimum necessary medium (MEM) (Logan, UT, United States). The Beyotime Cell Counting Kit-8 was purchased (Shanghai, China).
6
Transient Transfection and Analysis of HIV Env
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293FT cells (2×105) were transiently transfected with pHIV Env and tethered expression vectors by FuGENE HD in a 6-well culture plate. Cells were lysed with 60 µl RIPA lysis buffer (Thermofisher Scientific, MA, USA) at 48 h after transfection. After centrifugation at 20,400×g for 30 min at 4°C, the supernatant was collected and the protein concentration was determined by Pierce BCA protein assay (Pierce Biotechnology, Rockford, USA). Samples containing about 50 µg proteins were loaded into each well, electrophoresed (10% SDS-PAGE, Bio-Rad Ready Gel J) and transferred to a polyvinylidene fluoride membrane (Millipore, Immobilon-PSQ). The blot was probed with anti-gp120 polyclonal antibody (Fitzgerald, Concord, MA, USA), monoclonal anti-FLAG antibody (Sigma-Aldrich), or monoclonal antibody Chessie 8 [54] (link). Donkey anti-Goat IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, USA) or goat anti-Mouse IgG-HRP (Santa Cruz Biotechnology) was used as secondary antibodies. The blot was further treated with ECL Western Blot Kit (CWBIO, Beijing, China). Images were obtained with LAS3000 (Fujifilm, Tokyo, Japan).
7
Western Blot Analysis of Inflammasome Proteins
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Lysates were obtained by lysing the cell monolayer with phenylmethanesulfonyl fluoride (PMSF) and radioimmunoprecipitation assay (RIPA) lysis buffer (1:100). Total protein was separated in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to polyvinylidene difluoride (PVDF) membranes. Proteins were detected after labeling with specific primary antibodies [rabbit anti-NLRP3 (1:500, Bioss, China), rabbit anti-ASC polyclonal antibody (1:500, Bioss, China), rabbit anti-Caspase-1 (1:1000, Abcam, USA), rabbit anti cleaved Caspase1 (Arg317)/P10 (1:1000, Affinity, USA), rabbit anti-IL-1β antibody (1:1000, Abcam, USA), rabbit anti-BAX antibody (1:10000, Proteintech, USA), and rabbit anti-Bcl2 antibody (1:1500, Proteintech, USA)] followed by HRP-coupled secondary antibodies. The signals were detected by the addition of the chemiluminescent substrate (ECL Western Blot Kit, CWBIO, China). The protein amount was determined using Image J software. The level of GAPDH (1:1500, Proteintech, USA) was used to normalize the signal intensities.
8
SDS-PAGE and Western Blot Analysis
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Total protein was extracted and then separated with a 10% (w/w) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and the resulted proteins from the gel were blotted onto polyvinylidene difluoride (PVDF) membranes. The blot was blocked with 5% (w/w) skimmed milk for 2 h at room temperature and subsequently incubated overnight with the primary antibody at 4°C. Afterward, the blot was incubated with the antibody of horseradish peroxidase-conjugated anti-rabbit IgG (CWBio, Beijing, China) for 2 h. The immunoreaction was detected using the ECL Western Blot Kit (CW00495, CWBio, Beijing, China), and the ChemiDoc™ XRS imaging system (Bio-Rad Laboratories Inc., Hercules, CA, United States) was used to record the chemiluminescence on blots. The primary CBF1 antibody was ordered from GenScript Company (Nanjing, China).
9
Atrial Protein Expression Analysis
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Proteins from left atrial tissues were analyzed by western blotting. The atrial tissues were homogenized in radioimmunoprecipitation assay lysis buffer (Solarbio Institute of Biotechnology, Shanghai, China) and protein concentrations were determined using the Bradford Protein Assay Kit (BMG LABTECH, Offenburg, Germany). Solubilized protein was denatured in Lane Maker Loading Buffer (Cwbio, Beijing, China), separated by SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% or 8% gels and transferred to polyvinylidene difluoride filter membranes (Beyotime Institute of Biotechnology, Shanghai, China). Membranes were blocked with 5% nonfat dry milk in phosphate buffered saline (PBS) at room temperature. After 2 h, the membranes were incubated with rabbit phospho-PPARγ (S112) polyclonal antibody (1:1000; Elabscience, Wuhan, China) or with a rabbit polyclonal antibody to β-actin (1:1000; ComWin Biotech, Beijing, China) at 4℃ overnight. Membranes were incubated with secondary antibodies (1:1000; ZSGB-BIO, Beijing, China) for 2 h at room temperature. After extensive washing with phosphate buffered solution (PBST), bands were visualized with the ECL Plus western blotting detection system (ECL Western Blot Kit; CWBIO, Taizhou, China) and then quantified using Image J software (USA National Institute of Health, Bethesda, USA).
10
Western Blot Protein Quantification Protocol
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Cells were lysed in radioimmunoprecipitation assay buffer (Sigma) supplemented with a protease inhibitor cocktail (Roche). Equal amounts of protein extract were loaded onto gels for 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, the proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) and incubated in skimmed milk for 1 h at room temperature. The membrane was incubated with indicated primary antibodies at 4 °C overnight. The membrane was incubated with goat anti-rabbit IgG (Abcam) and then visualized using an ECL Western Blot Kit (CWBIO). Protein levels were quantified using Image J software (NIH, Bethesda, MD, USA). After the background was subtracted, the signal of each target band was normalized to that of the β-actin band. The antibody information is shown in Supplementary Table 1 .
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