Protein was extracted from cell samples with RIPA (Radio Immunoprecipitation Assay) lysate buffer containing 1% PMSF (Solarbio, Beijing, China). The protein concentration was determined with the BCA Kit (Beyotime, Shanghai, China). The proteins were separated with 10% SDS–polyacrylamide gel (Bio-Rad, Hercules, CA, USA) electrophoresis and transferred to polyvinylidine fluoride membranes. After the membranes were incubated with primary antibodies specific for CyclinD1, CDK2, PCNA, MyHC, MyoG, and β-actin (Abcam, Cambridge, MA, USA) and then the respective secondary antibodies, the membranes were exposed with ECL Plus (Solarbio, Beijing, China) and the images generated were captured with the ChemiDoc XRS+ system (Bio-Rad, Hercules, CA, USA).
Ecl plus
Manufactured by Solarbio
Sourced in China, United States
ECL Plus is a chemiluminescent detection reagent used in Western blotting techniques. It provides a sensitive and quantitative method for the detection of target proteins on membranes.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xrs system, by Bio-Rad (5 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (4 mentions)
Bca kit, by Beyotime (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Sds polyacrylamide gel, by Bio-Rad (2 mentions)
β actin, by Abcam (2 mentions)
Phenylmethylsulfonyl fluoride, by Wuhan Servicebio Technology (2 mentions)
Alphaview 3, by Bio-Techne (2 mentions)
Iseq00010, by Merck Group (2 mentions)
Ripa lysis buffer, by Wuhan Servicebio Technology (2 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (2 mentions)
Ripa lysis buffer, by Solarbio (2 mentions)
Mouse anti actin, by Wuhan Servicebio Technology (1 mentions)
Mouse anti acox1 sc 517306, by Santa Cruz Biotechnology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Wuhan Servicebio Technology (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Vegfa, by Abcam (1 mentions)
Ab254324, by Abcam (1 mentions)
Ab134175, by Abcam (1 mentions)
Ab179467, by Abcam (1 mentions)
19 protocols using ecl plus
1
Western Blot Analysis of Protein Expression
2
Adipocyte Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein of adipocyte samples was extracted by using RIPA lysis buffer supplemented with phenylmethylsulfonyl fluoride (Servicebio, Wuhan, China) (100:1). Proteins in cell lysates were separated by 10% SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (ISEQ00010, Millipore, Boston, MA, USA). The membranes were blocked with 5% nonfat milk for 1 h and then incubated with mouse anti-ACAA1 (sc-514051, Santa Cruz, Dallas, TX, USA), and mouse anti-ACOX1 (sc-517306, Santa Cruz, Dallas, TX, USA) antibodies at 4 °C overnight, followed by incubation with a secondary antibody conjugated with horse radish peroxidase (HRP) (GB23303, Servicebio) for 1 h at room temperature. Mouse anti-actin (Servicebio,) was used as the internal control. Signals were enhanced by ECL Plus (Solarbio, Beijing, China), and images were captured and analyzed by Photoshop CS6 and AlphaView 3.0 (Alpha Innotech, San Jose, CA, USA).
3
Western Blot Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from cells using a RIPA buffer (Solarbio) supplemented with PMSF (Servicebio) (100:1). Protein was separated on 10% SDS-PAGE gels. The proteins were transferred to PVDF membranesm, and then blocked with 5% non-fat milk for 2 h. The membranes were washed with PBST three times (5 min/time) and incubated with the primary antibodies (Abcam) at 4°C for overnight. Then the membranes were washed three times using PBST and incubated with secondary antibody conjugated with HRP (Abcam) for 1 h at room temperature. Signals were detected by ECL Plus (Solarbio). β-Actin was used as an internal control.
4
Western Blot Analysis of VEGFA and Apoptosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expressions of VEGFA protein and apoptosis-related genes were detected using Western blot. After different treatments for 48 h, the cultured cells were collected and lysed in RIPA (Beyotime), as were the thawed tissues. The protein concentration was determined by a BCA™ Protein Assay Kit (Pierce, Rockford, IL, USA). 20 μg amounts of samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Prior to the incubation of the specific primary antibodies overnight at 4 °C with gentle shaking, the blots were blocked in 5% nonfat milk. GAPDH on the same membrane was the loading control. After incubation of the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies, the proteins were visualized with ECL Plus (Solarbio, Beijing, China), and ImageJ was used to analyze the gray intensity of the bands. The primary antibodies were as follows: VEGFA (#46154, Abcam), Bcl-2 (#2872, CST), cleaved caspase 3 (#9661, CST), Bax (#2774, CST), p-AKT (#2965, CST) AKT (#9272, CST) and GAPDH (#5174, CST).
5
Examining Protein Expression in MTEC1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MTEC1 cells were inoculated into 6-well culture plates. When the cell fusion degree reached 60%, the overexpression vector, siRNA, miRNA, mimic and negative control were transfected into the cells. After 48 h, cell proteins were extracted using RIPA buffer and protease inhibitor mixture and then separated by 10% SDS-PAGE and immunoblotted using various antibodies according to standard western blot procedures. Rabbit anti-IGFBP5 (ab254324; Abcam, Cambridge, UK), rabbit anti-p53 (5395S; Cell Signaling Technology, Danvers, USA), rabbit anti-p21 (2947S; Cell Signaling Technology), rabbit anti-Bax (5023P; Cell Signaling Technology), rabbit anti-Cyclin B1 (ab55184; Abcam), rabbit anti-Cyclin D1 (ab134175; Abcam) and rabbit anti-β-actin (ab179467; Abcam) of 1:1000 dilution were incubated with protein blots at 4°C overnight. Subsequently, the secondary antibody (goat anti-rabbit) (A0208; Beyotime) of 1:1000 dilution was use to incubate with the blots at 37°C for 1 h. Finally, chemiluminescence detection was carried out using the ECL Plus (Solarbio, Beijing, China).
6
AMHR-2 Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were lysed in RIPA buffer, and the protein concentration was determined by BCA. Samples were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto PVDF membranes. Prior to incubation with primary antibodies overnight at 4°C with gentle shaking, the blots were blocked in 5% nonfat milk. The primary antibody used for this study was a rabbit polyclonal antibody against AMHR‐2 produced by Abcam, Cambridge, MA, USA (ab197148). GAPDH on the same membrane was the loading control. After incubation with horseradish peroxidase‐conjugated secondary antibodies, proteins were visualized with ECL Plus (Solarbio).
7
Western Blot Quantification of MAGE-A and SLC7A5
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed according to standard method. Proteins were extracted by RIPA lysis buffer (solarbio, life sciences). After denaturation, proteins were discreted by 10% SDS-PAGE electrophoresis and were transferred onto polyvinylidene fluoride (Millipore, Billerica, MA, USA). Mouse MAGE-A 6C1 antibody (Santa Cruz, USA), and Rabbit SLC7A5 antibody (Abcam, USA) were applied for immunoreactivity. The PVDF membranes were imaged using the ECL Plus (solarbio, life sciences).
8
Western Blot Analysis of Liver Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from liver tissue and cells using a RIPA lysis buffer supplemented with phenylmethyl sulfonyl fluoride (Servicebio, Wuhan, China) (100:1). Protein was separated on 10% SDS-PAGE gels. Separated protein was then transferred to polyvinylidene difluoride (PVDF) membranes (ISEQ00010, Millipore, Danvers, MA, USA) and membranes were then blocked with nonfat milk (5%) for 1 h. The membranes were washed with PBST three times (5 min/time) and incubated with the primary antibodies (Abcam) at 4 °C for overnight. Then the membranes were washed three times using PBST, and incubated with secondary antibody conjugated with HRP (GB23303, Servicebio, China) for one hour at room temperature. Signals were enhanced by ECL Plus (Solarbio) and images were captured and analyzed by Photoshop cs 6 and AlphaView 3.0 (Alpha Innotech, San Jose, CA, USA). β-Actin was used as an internal control.
9
Western Blot Analysis of Tumor Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins from tumor tissues and cells were extracted using RIPA lysis buffer (# R0020, Solarbio, Beijing, China), and protein concentrations were evaluated using BCA protein assay kit (# PC0020, Solarbio, Beijing, China). In total, proteins were separated by SDS-PAGE and then transferred onto PVDF membranes (# IPFL00010, Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk for 1 h and incubated with primary antibodies at 4°C overnight. After washing with TBST, secondary antibody incubations were performed at room temperature for 2 h. The bands were detected by chemiluminescence using ECL Plus (# PE0010, Solarbio, Beijing, China) according to the manufacturer’s instructions.
10
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from cell samples by using RIPA lysate buffer containing 1% PMSF (Solarbio, Beijing, China). The BCA Kit (Solarbio, Beijing, China) was used to measure the concentration of proteins in solution. The proteins were separated by 10% SDS- polyacrylamide gel (Bio-Rad, United States) electrophoresis and transferred to 0.22 μm nitrocellulose membranes (PALL, United States). After the membranes were incubated with primary antibodies specific for anti-cyclin D (cat. no. ab16663; dilution ratio = 1/200), anti-Bcl-2 (cat. no. ab692; dilution ratio = 1/200), anti-cyclin E (cat. no. ab33911; dilution ratio = 1/1000), anti-P27 (cat. no. ab32034; dilution ratio = 1/5000), anti-GAPDH (cat. no. ab8245; dilution ratio = 1/1000), and β-actin (cat. no. ab6276; dilution ratio = 1/5000) from Abcam (Cambridge, MA, United States) and secondary antibodies (dilution ratio = 1/5000; Solarbio, Beijing, China), the membranes were exposed with ECL Plus (Solarbio, Beijing, China). The images obtained were captured with the ChemiDoc XRS + system (Bio-Rad, United States).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!