Cellular proteins were collected 72 h after transfection and lysed with RIPA lysis buffer and phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology). A BCA Protein Assay kit (cat. no. P0010; Beyotime Institute of Biotechnology) was used to detect protein concentration. Protein samples (40 µg/lane) were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% skimmed milk in TBST buffer (TBS with 0.1% Tween-20) for 2 h at room temperature to avoid non-specific staining, then incubated with primary antibody overnight at 4˚C and HRP-conjugated secondary antibodies (cat. no. ZB-2301; OriGene Technologies, Inc.; 1:50,000) for 30 min at 37˚C. Primary antibodies used were as follows: PKN1 (cat. no. ab231038; Abcam; 1:500) and GAPDH (cat. no. sc-25778; Santa Cruz Biotechnology, Inc.; 1:5,000). Protein bands were visualized by enhanced ECL chemiluminescence (Beyotime Institute of Biotechnology). IOD values were measured with the Image-Pro Plus 6.0 system (Media Cybernetics, Inc.).
Enhanced chemiluminescence ecl
Manufactured by Beyotime
Sourced in China
Enhanced chemiluminescence (ECL) is a laboratory technique used to detect and quantify specific proteins in a sample. It relies on the emission of light produced by a chemical reaction to visualize the target proteins. ECL is commonly used in western blotting and other protein analysis methods.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Gapdh, by Abcam (1 mentions)
Image pro plus 6.0 system, by Media Cybernetics (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Occludin ab31721, by Abcam (1 mentions)
Phosphatase inhibitor, by Beyotime (1 mentions)
Sds page gel, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Jnk inhibitor sp600125, by Merck Group (1 mentions)
3 methyladenine 3 ma, by Merck Group (1 mentions)
Lc3 2, by Santa Cruz Biotechnology (1 mentions)
Becline 1, by Santa Cruz Biotechnology (1 mentions)
Lc3 1, by Santa Cruz Biotechnology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
6 protocols using enhanced chemiluminescence ecl
1
Western Blot Quantification of Cellular Proteins
2
Immunoblotting of Tight Junction Proteins
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IPEC-J2 cells were rinsed twice with ice-cold PBS and lysed by RIPA buffer (Invitrogen, USA) containing protease inhibitors, phosphatase inhibitor, and PMSF (Beyotime, Shanghai, China). The supernatant was collected, and a BCA protein assay kit (Beyotime, Shanghai, China) was used to quantify protein concentration. Cell supernatants were resolved on 10% SDS-PAGE gels (Beyotime, Shanghai, China), transferred on PVDF membranes (Millipore), and incubated with the corresponding primary antibodies overnight at 4°C (Claudin-1: ER1906-37, 1:1,000, Huabio; Occludin: ab31721, 1:1,000, Abcam; IL-17A: ER1902-37, 1:1,000, Huabio; beta-actin: 4967, 1:1,000, Cell Signaling). After multiple washing with TBST, the membranes were incubated with secondary antibodies (1:5,000, Huabio, Hangzhou, China) at room temperature for 1 h. Detection was performed by enhanced ECL chemiluminescence (Beyotime, Shanghai, China) and captured by Imaging System. The bands’ intensity was analyzed by ImageJ.
3
Propranolol's Apoptotic and Autophagic Effects
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Propranolol was obtained from the Chinese materials research center (Beijing, China). Fetal bovine serum (FBS) was got from Gibco, Gaithersburg, MD, USA). N-Acetyl-L-cysteine (NAC), 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Hoechst 33258 staining, and Annexin V-PE Apoptosis Detection Kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The enhanced chemiluminescence (ECL) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). JNK inhibitor (SP600125) and 3-MA (3-Methyladenine) were obteined from sigma (St. Louis, MI, USA). Antibodies against Cyclin B1, phospho-cdc2, cdc2, JNK, phosphorylated JNK, and p21 were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against Cdk1, Bcl-2, cleavage caspase-3, cleavage caspase -8, cleavage caspase -9, BAX, LC3-I, LC3- II, Becline-1, p62, GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other common chemicals and buffers were from Boster (Wuhan, China).
4
Protein Expression Analysis by Western Blot
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Protein was extracted and the concentration of protein was measured with the BCA protein assay kit. After being separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis
(SDS-PAGE), proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and then was blocked with 5% non-fat milk in tris-buffered saline with
tween-20 (TBST) for 1 h. The membranes were incubated with the primary antibodies against EZH2 (1:500, ABclonal Biotechnology Co., Ltd.) and H3K27Me3 (1:500, ABclonal Biotechnology Co.,
Ltd.), HistoneH3 (1:500, ABclonal Biotechnology Co., Ltd.) and GAPDH (1:500, ABclonal Biotechnology Co., Ltd.) at 4°C overnight and then were incubated with the HRP-labeled goat anti-rabbit
IgG secondary antibody (1:10,000, Beyotime Biotech Co., Ltd.) for 40 min at 37°C. Enhanced chemiluminescence (ECL; Beyotime Biotech Co., Ltd.) was added to visualize the bands. The optical
density was analyzed by using Tanon Image software. HistoneH3 and GAPDH were used as the internal reference.
(SDS-PAGE), proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and then was blocked with 5% non-fat milk in tris-buffered saline with
tween-20 (TBST) for 1 h. The membranes were incubated with the primary antibodies against EZH2 (1:500, ABclonal Biotechnology Co., Ltd.) and H3K27Me3 (1:500, ABclonal Biotechnology Co.,
Ltd.), HistoneH3 (1:500, ABclonal Biotechnology Co., Ltd.) and GAPDH (1:500, ABclonal Biotechnology Co., Ltd.) at 4°C overnight and then were incubated with the HRP-labeled goat anti-rabbit
IgG secondary antibody (1:10,000, Beyotime Biotech Co., Ltd.) for 40 min at 37°C. Enhanced chemiluminescence (ECL; Beyotime Biotech Co., Ltd.) was added to visualize the bands. The optical
density was analyzed by using Tanon Image software. HistoneH3 and GAPDH were used as the internal reference.
5
Molecular Mechanisms of Neuronal Apoptosis
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Rabbit anti-NDRG4 antibody (cat. no. 9039), anti-Bax antibody (cat. no. 14796), anti-Bcl-2 antibody (cat. no. 2876), anti-caspase-3 antibody (cat. no. 9662) (Cell Signaling Technology, USA); Rabbit anti-BDNF antibody (cat. no. ab108319), anti-c-Fos antibody (cat. no. ab134122) and anti-β-actin (cat. no. 4044) (Abcam, USA); TRIzol buffer, radioimmune precipitation buffer (RIPA), 4′6-diamidino-2-phenylindole (DAPI), bicinchoninic acid protein assay kit (BCA), enhanced chemiluminescence (ECL) (Beyotime Technology, Shanghai, China); anti Alexa Fluor 647-goat anti-rabbit IgG (Abcam, USA); anti-rabbit FITC tagged secondary antibody (Abcam, USA); terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) kit (Roche, Basel, Switzerland); horseradish peroxidase-conjugated anti-rabbit secondary antibody (cat. no. sc-2004, Santa Cruz Biotechnology Inc, USA).
6
Western Blot Analysis of Stat3 Protein
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The RIPA buffer containing 1% PMSF was used to lyse the cells to obtain the protein samples. BCA protein assay kits (Beyotime, Beijing, China) were applied for the determination of the protein concentration. Proteins were separated on 8% SDS-PAGE gels and electrophoretically transferred to the membranes of PVDF (0.22 µm pore size; Millipore, Boston, MA, USA). The PVDF membranes were blocked with 5% BSA at room temperature for one hour and then incubated with an antibody against Stat3 (1:2000; 79D7, CST, Danvers, MA, USA) at 4 °C overnight. Subsequently, the blots were incubated with a secondary antibody (1:5000; Beyotime, Beijing, China) for 2 h at room temperature. Immunoreactivity was detected using Enhanced Chemiluminescence (ECL) (Beyotime, Beijing, China) and Quantity One software (Bio-Rad, Hercules, California, USA). Gapdh (1:2000; D16H11, CST, Danvers, MA, USA) was utilized as an internal control. The experiment was performed in 3 replicates.
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