After the completion of treatment, around 2 × 106/ml cells were disintegrated in one condition. After that, BCA Protein Assay Kit (Beyotime Company, Shanghai, China) was used to calculate the protein concentration. It is evaluated that whole cell lysates were then electrophoresed in 8–12 percent SDS-PAGE before being put into polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). The blots were then envisioned with the help of Odyssey CLx imaging system by ECL reagents (Yeasen Biotech Co., Ltd., Shanghai, China).
Ecl reagent
Manufactured by Yeasen
Sourced in China, United States
ECL reagent is a laboratory product used for the detection and visualization of proteins in Western blot analysis. It generates a luminescent signal that can be detected and quantified by a luminometer or X-ray film. The reagent contains the necessary components for the chemiluminescent reaction, allowing for sensitive and specific detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Bca kit, by Beyotime (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (2 mentions)
Ab207612, by Abcam (1 mentions)
Ac026, by ABclonal (1 mentions)
T40044, by Abmart (1 mentions)
Goat anti mouse igg h l, by Yeasen (1 mentions)
T40056, by Abmart (1 mentions)
Peroxidase conjugated goat anti rabbit igg h l, by Yeasen (1 mentions)
Bradford reagent, by Beyotime (1 mentions)
Sybr green qpcr mix, by Yeasen (1 mentions)
Trizol rna purification kit, by Thermo Fisher Scientific (1 mentions)
β actin antibody, by Proteintech (1 mentions)
Pvdf western blotting membrane, by Roche (1 mentions)
17 protocols using ecl reagent
1
Protein Quantification and Western Blot
2
Cerebral Cortex Protein Analysis
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Total proteins were extracted from the right cerebral cortex in the ischemic penumbra and quantitated using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein were separated with 8–12% SDS-PAGE gels and electrically transferred to PVDF membranes (Millipore, Burlington, MA, USA). The membranes were incubated with primary antibodies against Bcl-2 (1:1000, T40056, Abmart, Shanghai, China), Beclin1 (1:2000, ab207612, Abcam, Cambridge, UK), Bax (1:10,000, 50599-2-Ig, Proteintech, Wuhan, China), caspase3 (1:1000, T40044, Abmart, Shanghai, China), LC3B (1:2000, T40044, Abcam, Cambridge, UK), ZO-1(1:2000, 21773-1-AP, Proteintech, Wuhan, China), Occludin (1:10,000, 66378-1-Ig, Proteintech, Wuhan, China), β-actin (1:100,000, AC026, Abclonal, Wuhan, China), and GAPDH (1:10,000, 60004-1-Ig, Proteintech, Wuhan, China) at 4 °C overnight. They were then incubated in Peroxidase-Conjugated Goat Anti-Rabbit IgG(H+L) or Goat Anti-Mouse IgG(H+L) (Yeasen, Shanghai, China) diluted at 1:10,000 for 1 h at room temperature. After chemiluminescence detection had been performed using ECL reagents (Yeasen, Shanghai, China) and a Fusion Edge Multi-function Imaging System (Vilber, Collégien, France), the relative optical densities of the protein bands were analyzed using ImageJ software.
3
Protein Quantification and Western Blot Analysis
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After the treatment, the cells were lysed using an RIPA lysis buffer and the protein content was determined using Bradford reagent (Beyotime Biotechnology, Shanghai, China). Afterwards, Western blotting was performed. The samples were separated using 10% (w/v) sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis to separate the proteins. Electrophoresis was performed at 80 V for 2 h. The proteins were transferred to PVDF membranes at 100 V for 1 h. Afterwards, the membrane was blocked with 5% (w/v) non-fat dry milk at room temperature for 2 h, the corresponding antibody was added and incubated overnight at 4◦C, and it was incubated with the secondary antibody for 2 h at room temperature. Finally, the proteins were visualized using ECL reagents (YEASEN, Shanghai, China) and a gel imager (Syntyos, Cambridge, MA, USA).
4
Comprehensive RNA Expression Analysis
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Leibovitz's L-15 cell culture medium (Cat. 11415114), fetal bovine serum (FBS), penicillin-streptomycin (Cat. 15140122), lipofectamine 3000, and TRIzol RNA purification kit (Cat. 12183555) were purchased from ThermoFisher Scientific. First-strand cDNA synthesis kit with genomic DNA digester, ECL reagents, and qPCR SYBR Green Mix were all purchased from Yeasen Biotech. ADAM28 polyclonal antibody (Cat. 22234-1) and β-actin antibody (Cat. 20536-1) were obtained from Proteintech Group. The primers of the indicated target genes for RT-PCR were produced by Invitrogen. Gemcitabine was from TCI, and other chemical reagents are analytical grade and were provided by Aladdin.
5
Western Blot Analysis and IP Protocol
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Cells were lysed using cell lysis buffer for Western blot analysis and IP (Beyotime, Shanghai, China, P0013) supplemented with 1% PMSF (Beyotime, Shanghai, China, ST505). BCA Protein Assay Kit (Beyotime, Shanghai, China, P0010) was used to measure protein concentration. 12% SDS-PAGE was used to resolve proteins. Proteins were electrophoretically transferred onto PVDF Western blotting membranes (Roche, Mannheim, Germany, 3010040001). Proteins in the membranes were analyzed by enhanced chemiluminescence (ECL) reagents (Yeasen, Shanghai, China, 36208ES76) as described previously [42 (link)].
Primary antibodies were anti-Flag (mouse) (Abmart, Shanghai, China, M20008H), anti-Tubulin (Rabbit) (Beyotime, Shanghai, China, AF0001), and anti-BmCyclin B (Rabbit), which was previously prepared in our laboratory. HRP-labeled goat anti-mouse (or anti-rabbit) IgG (H + L) was applied as secondary antibody. Protein markers were Blue PlusTM protein marker (TransGen, Beijing, China, DM111-01) and EasySeeTM Western Marker (TransGene Biotech, Beijing, China, DM201-02).
Primary antibodies were anti-Flag (mouse) (Abmart, Shanghai, China, M20008H), anti-Tubulin (Rabbit) (Beyotime, Shanghai, China, AF0001), and anti-BmCyclin B (Rabbit), which was previously prepared in our laboratory. HRP-labeled goat anti-mouse (or anti-rabbit) IgG (H + L) was applied as secondary antibody. Protein markers were Blue PlusTM protein marker (TransGen, Beijing, China, DM111-01) and EasySeeTM Western Marker (TransGene Biotech, Beijing, China, DM201-02).
6
Protein Extraction and Co-Immunoprecipitation Protocol
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Cells were lysed in lysis buffer (1%SDS and 30 μM Tris-HCl [pH6.8]). Total proteins (10 μg) were separated on SDS-PAGE and then transferred onto PVDF membranes (Millipore). After blocking using 5% nonfat milk, membranes were incubated with the gene-specific primary antibodies, then HRP-conjugated secondary antibody (Jackson ImmunoResearch), and visualized using ECL reagents (YEASEN). Antibodies used in this study were listed in Supplementary Table 3.
For co-immunoprecipitation (co-IP), 24 h after transfection, cell lysates were lysed in 1% Triton lysis buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 150 mM NaCl and 1% Triton X-100) containing protease and phosphatase inhibitors. The lysates were subjected to co-IP using specific antibody-conjugated agarose (Sigma) for 2 h. After extensive washes, immunoprecipitated proteins were separated on SDS-PAGE, transferred to PVDF membranes and detected by Western blotting with appropriate antibodies.
For endogenous and semi-endogenous immunoprecipitation, cell lysates were lysed in 1% Triton lysis buffer and sonicated for a short time. Immunoprecipitation was carried out with Flag-conjugated agarose or anti-RNF214 (J044) antibody and protein A Sepharose (GE Healthcare). After incubation at 4 °C overnight and several washes, precipitated proteins were eluted with 0.1 M Glycine (pH 3.0) and separated by SDS-PAGE.
For co-immunoprecipitation (co-IP), 24 h after transfection, cell lysates were lysed in 1% Triton lysis buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 150 mM NaCl and 1% Triton X-100) containing protease and phosphatase inhibitors. The lysates were subjected to co-IP using specific antibody-conjugated agarose (Sigma) for 2 h. After extensive washes, immunoprecipitated proteins were separated on SDS-PAGE, transferred to PVDF membranes and detected by Western blotting with appropriate antibodies.
For endogenous and semi-endogenous immunoprecipitation, cell lysates were lysed in 1% Triton lysis buffer and sonicated for a short time. Immunoprecipitation was carried out with Flag-conjugated agarose or anti-RNF214 (J044) antibody and protein A Sepharose (GE Healthcare). After incubation at 4 °C overnight and several washes, precipitated proteins were eluted with 0.1 M Glycine (pH 3.0) and separated by SDS-PAGE.
7
Western Blot Protein Analysis Protocol
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Cells were generally lysed with 4% SDS lysis buffer containing a protease inhibitor cocktail and benzonase unless otherwise stated. Cell lysates were clarified by centrifugation at 12,000g for 20 min and quantified by the BCA assay (Pierce). Equal amounts of cell lysates were diluted with 4× reducing SDS-loading buffer and β-mercaptoethanol. The samples were heated for 10 min at 95 °C and loaded onto 4 to 20% Bis-Tris gels (Genscript) for SDS-PAGE separation. Gels were transferred onto nitrocellulose membranes using the Bio-Rad Trans-Blot Turbo Transfer System (25 V, 30 min). After blocking with 5% nonfat milk in PBST (0.05% Tween-20 in PBS) for 30 min at room temperature, the membranes were incubated overnight with primary antibodies at 4 °C. Following PBST washes three times, appropriate secondary antibodies were added, and the membranes were developed with ECL reagents (Yeasen). Membranes were imaged using a ChemiDoc MP Imager (Bio-Rad). The protein band of interest was quantified using ImageLab software (Bio-Rad).
8
Osteogenic Protein Expression Analysis
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AS fibroblasts were lysed with RIPA buffer (Beyotime Shanghai, China) to extract total protein. After quantified using BCA Kit (Beyotime), equal amounts of proteins were separated with SDS-PAGE gel and transferred onto PVDF membranes (Millipore). Then, the membranes were blocked with 5% non-fat milk and incubated with antibodies against runt-related transcription factor 2 (RUNX2; 1:1,000, ab76956, Abcam, Cambridge, MA, United States), osteocalcin (OCN; 1:500, ab93876, Abcam), osteoprotegerin (OPG; 1:1,000, ab73400, Abcam), SOST (1:1,000, ab85799, Abcam) or β-actin (1:5,000, ab8227, Abcam) at 4°C overnight. After incubated with secondary antibodies against Goat Anti-Rabbit IgG (1:10,000, ab205718, Abcam) or Goat Anti-Mouse IgG (1:5,000, ab205719, Abcam), protein signals were visualized using enhanced chemiluminescence (ECL) reagent (Yeasen). The experiment was performed three biological replicates.
9
Western Blot Analysis of Macrophage Proteins
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Western blot was performed as previously described [19 (link)]. In brief, the protein extracted from the macrophages was degenerated, electrophoresed, and transferred to the polyvinylidene fluoride membrane (Millipore). Subsequently, the membrane was blocked using 5% non-fat milk and incubated with primary antibodies—rabbit anti-PFKFB3 (Abcam), iNOS (inducible nitric oxide synthase) (Proteintech), CD206, Jak1, P-Jak1, Jak2, P-Jak2, Jak3, P-Jak3, Stat 1, P-stat1 (Y701), P-stat1 (Y727) (all from CST technology), and mouse anti-β-actin (Abcam)—overnight at 4 °C. On the following day, membranes were washed and incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (Jackson Laboratory) and detected with an ECL reagent (Yeasen, Shanghai, China) using the chemiluminescence system (Tanon, Shanghai, China). The grayscale of the detected bands was assessed by ImageJ software (National Institutes of Health), and the results were expressed as fold changes after normalizing to β-actin.
10
Exploring Apoptotic Signaling Pathways in HK2 Cells
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HK2 cells were lysed in RIPA buffer (Beyotime), and the cell lysates were centrifugated at 14,000 g for 1 min. The supernatants were obtained, and protein concentration was detected with a BCA kit (Amyjet, Wuhan, China). A 20 µg protein sample was separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane (Solarbio). The membrane was blocked in 5% bovine serum albumin (Solarbio). Subsequently, the membrane was incubated with antibody against B-cell lymphoma-2 (Bcl-2) (ab59348, 1 : 500 dilution, Abcam), Bcl-2-associated X (Bax) (ab263897, 1 : 2000 dilution, Abcam), cleaved caspase 3 (C-caspase 3) (ab2302, 1 : 1000 dilution, Abcam), IRAK1 (ab238, 1 : 2000 dilution, Abcam), or GAPDH (ab245355, 1 : 5000 dilution, Abcam) overnight and IgG labeled by horseradish peroxidase (HRP) (ab6721, 1 : 8000 dilution, Abcam) for 2 h. Next, the blots were interacted with ECL reagent (Yeasen), and then examined by Image J v1.8 software (NIH, Bethesda, MD, USA). GAPDH functioned as a loading reference, and relative protein expression was normalized to the control group.
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