Total proteins from the HaCat cells in 6-well plates were extracted using RIPA lysis buffer with 2% PMSF and phosphatase inhibitor (Cat # P0013K, Beyotime). The experiments were performed as previously described (20 (link)). The extracted proteins were separated using 10%SDS electrophoresis before transfer onto a nitrocellulose membrane. The membrane was separately probed by incubation with the respective primary antibody (c-MYB, Cat #17800-1-AP, Proteintech) overnight at 4°C, followed by incubation with horseradish peroxidase-labeled secondary antibody for 2 h at 37°C. Enhanced chemiluminescence reagent (Cat # MA0186, Meilunbio) was then added to the blots, and the bands were analyzed using ImageJ software (NIH, USA).
Enhanced chemiluminescence reagent
Manufactured by Meilun
Sourced in United States
Enhanced chemiluminescence reagent is a laboratory product that generates a luminescent signal when combined with a target analyte. It is designed to be used in various biochemical assays, such as Western blotting and ELISA, to detect and quantify the presence of specific proteins or other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor, by Beyotime (2 mentions)
C myb, by Proteintech (1 mentions)
Pappa2, by Abcam (1 mentions)
Bradford reagent, by Solarbio (1 mentions)
Caspase 1, by Proteintech (1 mentions)
Il 1β, by Proteintech (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by ABclonal (1 mentions)
Krt17, by Proteintech (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Proteintech (1 mentions)
Claudin 1, by Proteintech (1 mentions)
Icam2, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Bcl2 associated x (bax), by ZenBio (1 mentions)
N cadherin, by ZenBio (1 mentions)
Snail1, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Bcl 2, by Proteintech (1 mentions)
6 protocols using enhanced chemiluminescence reagent
1
Quantification of c-MYB in HaCat Cells
2
Quantitative Analysis of Placental Proteins
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Total proteins extracted from JEG-3 cells or JEG-3-ORGs were lysed in lysis buffer containing a protease inhibitor mixture and quantified using Bradford reagent (Solarbio, China). The lysates were separated by SDS-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. The membranes were incubated overnight at 4°C with primary antibodies against human HLA-G (1,1,000, Abcam, UK), PAPPA2 (1,1,000, Abcam, UK), and β-tubulin (1,5,000, proteintech, China); they were incubated subsequently with horseradish peroxidase-conjugated rabbit or mouse secondary antibodies (1,10,000, Abcam, UK). After treatment with enhanced chemiluminescence reagent (Meilunbio, China), a ChemiDoc image analyzer was used for densitometric analysis. β-tubulin served as the loading control.
3
Western Blot Analysis of ECM Proteases
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The experimental procedures were performed as our published paper described [34 (link)]. The extracted proteins were separated using 10% SDS electrophoresis before transfer onto a nitrocellulose membrane. The blots were cut prior to hybridisation with antibodies during blotting. The membrane was separately probed with the respective primary antibody (MMP2, Cat# A11144; MMP9, Cat# A0289, ABclonal; caspase-1, Cat# 22,915–1-AP, Proteintech; and IL-1β, Cat# 16,806–1-AP; Proteintech) for 8 h at 4 ºC followed by incubation with horseradish-peroxidase-labeled secondary antibody for 2 h at 37 ºC. Enhanced chemiluminescence reagent (Cat# MA0186, Meilunbio) was then added to the blots and the bands were analyzed using ImageJ software (NIH, USA).
4
Protein Extraction and Western Blot Analysis
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Total proteins from HEK and HaCaT cells in 6-well plates and tissues were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer containing 2% phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor (Cat # P0013K, Beyotime). Proteins were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. Protein probing was performed by overnight membrane incubation at 4 °C with primary antibodies (KRT17, Cat #18502-1-AP, Proteintech, RRID: AB 10644296). The membrane was then washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (Cat# AS014, ABclonal, RRID:AB 2769854) for 2 hours. Blots were developed using enhanced chemiluminescence reagent (Cat # MA0186, Meilunbio), and band intensities were analyzed using ImageJ software (NIH, USA).
5
Quantitative Protein Expression Analysis
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The extracted proteins were separated using 10% SDS electrophoresis before transfer onto a nitrocellulose membrane. The membrane was separately probed with a respective primary antibody (MMP-2, Cat # A11144; TIMP-2, Cat # A1558; MMP-9, Cat # A0289; TIMP-1, Cat # A1389, ABclonal) for 8 h at 4°C followed by incubation with horseradish peroxidase-labeled secondary antibody for 2 h at 37°C. Enhanced chemiluminescence reagent (Cat # MA0186, Meilunbio) was then added to the blots and the bands were analyzed using ImageJ software (NIH, USA).
6
Western Blot Analysis of Protein Expression
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To extract cellular proteins, a lysis buffer (Thermo Fisher Scientific) was employed. A BCA protein assay kit (Beyotime) was used to determine the protein concentration. Subsequently, equal amounts of the protein were separated by SDS-PAGE, and then transferred onto polyvinylidene difluoride membranes. These membranes were blocked using 5% skimmed milk and incubated overnight at 4 ℃ with primary antibodies targeting specific proteins, including ICAM2 (Cell Signaling Technology), GAPDH (Proteintech), BAX (ZENBIO), BAD (ZENBIO), BCL2 (Proteintech), E-cadherin (Proteintech), N-cadherin (ZENBIO), Claudin 1 (Proteintech), Snail1 (Proteintech), B-catenin (Abmart), HA (Proteintech), and RDX (ZENBIO). After washing, the membranes were incubated with secondary antibodies (Proteintech) for 1 h at room temperature. They were then visualized using an enhanced chemiluminescence reagent (Meilunbio) and captured using a ChemiDoc Touch imaging system (Bio-Rad).
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