Tissue samples were conserved in liquid nitrogen; total protein was extracted and determined using a BCA Protein Assay Kit according to the manufacturer's instruction. Equal amounts of protein (10 μg) for each sample were resolved by 10% acrylamide-SDS-PAGE. The samples were subsequently transferred to polyvinylidene difluoride membranes (PVDF; Millipore, Billerica, MA). Primary antibodies (rabbit anti-ADAMTS-7, 1 : 3,000, Abcam Biotechnology Co., Ltd., London, UK; rabbit anti-COMP, 1 : 3,000, Abcam Biotechnology Co., Ltd., London, UK), and a goat anti-rabbit immunoglobulin- (IgG-) horseradish peroxidase (HRP) secondary antibody (1 : 2,500; Beijing Golden Bridge Biotechnology Co., Ltd., Beijing, China) were applied. Equal amounts of protein loading were confirmed by reprobing the membranes with the rabbit anti-GAPDH-HRP antibody (1 : 5,000, Abcam Biotechnology Co.). Protein bands were detected using a FluorChem E Chemiluminescent Western Blot Imaging System (Cell Biosciences, Santa Clara, CA) and quantified by densitometry analysis using ImageJ software (National Institutes of Health, USA).
Fluorchem e chemiluminescent western blot imaging system
Manufactured by Cell Biosciences
Sourced in United States
The FluorChem E Chemiluminescent Western Blot Imaging System is a laboratory instrument designed for the detection and analysis of chemiluminescent signals in Western blot experiments. It provides high-quality imaging capabilities for researchers to visualize and quantify protein expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Rabbit anti comp, by Abcam (1 mentions)
Phosphate buffered saline (pbs), by Wuhan Servicebio Technology (1 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Tween 20, by Solarbio (1 mentions)
Gapdh, by Proteintech (1 mentions)
Enhanced chemiluminescence kit, by Cytiva (1 mentions)
β actin antibody, by Cell Signaling Technology (1 mentions)
Phospho stat3, by Abcam (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Stat3, by Abcam (1 mentions)
Beclin 1, by Abcam (1 mentions)
Ciap2, by Abcam (1 mentions)
Ciap1, by Abcam (1 mentions)
X linked inhibitor of apoptosis protein xiap, by Santa Cruz Biotechnology (1 mentions)
β actin, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
7 protocols using fluorchem e chemiluminescent western blot imaging system
1
Western Blot Analysis of ADAMTS-7 and COMP
2
Western Blot Protein Expression Analysis
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The protein expression levels were determined by staining with primary antibodies and relevant secondary (1:5000, Zhongshanjinqiao, China) antibodies. The primary antibodies [PARP, #9532; γ-H2AX, #9718; TATA-binding protein (TBP), #44059; H3, #4499; AceH3(K27), #8173, cell signaling technology, Herts, United Kingdom; YAP1, #13584-1-AP, Proteintech, China] were diluted at 1:1000 in 5% fat-free milk phosphate-buffered saline (PBS, Servicebio, China) with 0.05% TWEEN 20 (Solarbio, China) (PBST). Anti-β-actin (1:1000, #ZM-0003SMA, Zhongshanjinqiao, China) was used as the loading control of cell lines, and GAPDH (1:1000, #60004-1-Ig, Proteintech, Wuhan, China) was used as the loading control of primary cells from the relapsed FLT3-ITD+ AML patients. The protein bands were visualized using a FluorChem E Chemiluminescent Western Blot Imaging System (Cell Biosciences). The bidimensional optical densities of proteins on the films were quantified and analyzed with Quantity One software (ImageJ), and the relative values were calculated by comparing with the loading control.
3
Analyzing Autophagy-related Protein Levels
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Western blotting was performed according to the standard protocols, using antibodies against human LC3B (#3868, Cell Signaling, Danvers, MA, USA, 1:1000), p62 (#16177, Cell Signaling, 1:1000), STAT3 (#ab68153, Abcam, Cambridge, MA, USA 1:1000), phospho-STAT3(#ab76315, Tyr705; Abcam, 1:1000), beclin1 (#ab207612, Abcam, 1:1000), Bcl-2 (#4223, Cell Signaling, 1:1000), and β-actin antibody (#4970, Cell Signaling, 1:5000). The bands were visualised using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA) on FluorChem E Chemiluminescent Western Blot Imaging System (Cell Biosciences, Santa Clara, CA, USA). Cell Signaling Technology, Inc. (Danvers, MA, USA).
4
Western Blot Analysis of Apoptosis Markers
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Total protein from JEG3 and JEG3 tumors from different treatment groups was run in PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel and transferred to a PVDF (polyvinylidene fluoride) membrane and probed with the following primary antibodies, for 12 h, at 4 °C: caspase-3, caspase-9, p53, bax, bcl-2, AIF, cIAP 1 and cIAP 2 (Abcam Inc., Cambridge, MA, USA, 1:1000), X-linked inhibitor of apoptosis protein (XIAP) (Santa Cruz Biotechnology Inc., USA, 1:1000), poly (ADP-ribose)-polymerases (PARPs), and β-actin (Abcam Inc., Cambridge, MA, USA, 1:1000). Then, the blots were incubated with HRP (horseradish peroxidase)-conjugated goat anti-rabbit secondary antibody for 1 h and washed. Following detection with FluorChem E Chemiluminescent Western Blot Imaging System (Cell Biosciences, Santa Clara, CA, USA), the blots were quantified by densitometry analysis, using Image J software (National Institutes of Health, Bethesda, MD, USA). All Western blot data shown are representative of at least three independent experiments.
5
Protein Extraction and Western Blot Analysis
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Cells were lysed in a protein solubilization buffer. Protein extracts were prepared with a Total Protein Extraction Kit according to the manufacturer’s instructions (BestBio, Shanghai, China). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Millipore). β-actin or GAPDH served as a loading control. Primary antibodies included anti-GAPDH, anti-β-actin (ZSGB-BIO, China), anti-TNFAIP8, anti-Rac1 (Proteintech), anti-Flag (Sigma), as well as anti-ERK1/2, anti-p-ERK1/2, anti-MEK, and anti-p-MEK (CST). Protein bands were visualized using a FluorChem E Chemiluminescent Western Blot Imaging System (Cell Biosciences).
6
Quantitative Western Blot Analysis
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Protein was extracted and determined using a bicinchoninic acid (BCA) Protein Assay Kit (Sangon Biotech Co., Ltd, Shanghai, China). Samples with equal amounts of total protein (10 µg) were separated using polyacrylamide gel electrophoresis with 5% stacking and 12% separating gels. Proteins were subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) and stained with primary antibodies (goat anti-CDNF, 1∶1000, R & D Systems, Inc., Minneapolis, MN) and a rabbit anti-goat immunoglobulin (IgG)-horseradish peroxidase (HRP) secondary antibody (1∶30,000, Cell Signaling Technology, Danvers, MA). Loading of equal amounts of protein was confirmed by reprobing the membranes with a mouse anti-β-actin -HRP antibody (1∶10,000, Abcam, Cambridge, UK). Protein bands were detected using a FluorChem E Chemiluminescent Western Blot Imaging System (Cell Biosciences, Santa Clara, CA) and quantified by densitometry analysis using ImageJ software (National Institutes of Health, Bethesda, MD).
7
Western Blot Analysis of Apoptosis Regulators
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Forty‐eight hours after transfection, the cells were lysed in ice-cold radio immunoprecipitation assay (RIPA) buffer for 30min, and the protein concentration was quantified using Bio-Rad protein assay reagent (Bio-Rad, CA, USA). Thirty μg protein samples were loaded and separated on 10% SDS–polyacrylamide gels, and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were first blocked with 5% non-fat skim milk for 2h, then incubated with anti-Bcl-w (1:1000; Cell Signaling Technology, Beverly, MA, USA), anti-Survivin (1:1000; Abcam, Cambridge, Massachusetts, USA) or anti-β-actin rabbit monoclonal antibody (1:1000; Cell Signaling Technology) overnight at 4°C, followed by incubation with HRP-labeled secondary antibody (1:5000; Cell Signaling Technology) for 1h at room temperature. Finally, the bands were visualized using chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA) on FluorChem E Chemiluminescent Western Blot Imaging System (Cell Biosciences, Santa Clara, CA, USA).
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