Log In Sign up
The largest database of trusted experimental protocols

Westernbright ecl

Manufactured by Advansta
Visit Supplier
Sourced in United States, China, Canada

WesternBright ECL is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. It produces a luminescent signal upon interaction with the horseradish peroxidase (HRP) enzyme, which is typically conjugated to secondary antibodies in Western blotting experiments.

Automatically generated - may contain errors

Lab products found in correlation

Pvdf membrane, by Merck Group (22 mentions) Trans blot turbo system, by Bio-Rad (16 mentions) Hrp conjugated anti rabbit igg or anti mouse igg, by Abbkine (10 mentions) Bca protein assay kit, by Vazyme (10 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (9 mentions) Chemidoc mp imaging system, by Bio-Rad (8 mentions) Ripa buffer, by Beyotime (7 mentions) Pvdf membrane, by Thermo Fisher Scientific (7 mentions) Trans blot turbo transfer system, by Bio-Rad (6 mentions) Image lab software, by Bio-Rad (6 mentions) Bca protein assay kit, by Beyotime (6 mentions) β actin, by Merck Group (6 mentions) Phosstop, by Roche (6 mentions) Quantity one software, by Bio-Rad (6 mentions) Trans blot sd semi dry transfer cell, by Bio-Rad (5 mentions) Western bright peroxide, by Advansta (5 mentions) Protease inhibitor cocktail, by Roche (5 mentions) Nitrocellulose membrane, by Bio-Rad (5 mentions) Nitrocellulose membrane, by Cytiva (5 mentions) Hrp conjugated secondary antibody, by Promega (5 mentions)

232 protocols using westernbright ecl

1

Detailed Western Blot Protocol

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Western blot was performed as described previously25 (link). In brief, cells were lysed and cell debris was removed by centrifugation at top speed for 5 min at 4 °C. Proteins were resolved by 12% or 15% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked and sequentially incubated with corresponding primary and secondary antibodies with washing steps in between. Immunoblots were visualized by chemiluminescence using WesternBright ECL (Advansta). Unprocessed versions of original immunoblots can be found in Supplementary Figs. S6, S7, and S8. Several blots were cut prior to antibody hybridization for the simultaneous detection of different target proteins in the same sample.
Wong K.M., Song J, & Wong Y.H. (2021). CTCF and EGR1 suppress breast cancer cell migration through transcriptional control of Nm23-H1. Scientific Reports, 11, 491.
+ Open protocol
+ Expand
2

Quantitative Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Gel electrophoresis was in Nu-Page 4-12% gradient Bis-Tris NuPage gels (Life Technologies) followed by transfer to nitrocellulose. Mouse pan-specific antibody 9A7 (gift of Dr. Maureen McEnery, Case Western Reserve University, Cleveland, OH) was employed to detect the total amount of alpha subunit isoforms. Monoclonal antibodies 6F (Developmental Studies Hybridoma Bank) or M17-P5-F11 (gift of Dr. W. James Ball, University of Cincinnati) were used to detect human α1 and β2 subunits of Na,K-ATPase, respectively. α3 subunit of Na,K-ATPase was detected with the goat peptide-directed antibody C16 (Santa Cruz Biotechnology). Actin-HRP antibody was from Santa Cruz Biotechnology. Secondary antibodies were HRP-conjugated, and final detection was with chemiluminescence (WesternBright ECL, Advansta, CA). An LAS 4000 imager (GE Healthcare Life Sciences) with ImageQuant software was used for the analysis of Western blots. All analyses were performed with a minimum of 3 biological replicates, and actin was used as a loading control for all samples for quantification. When protein induction is large compared to the basal level, the accuracy of blot quantification is limited by the accuracy of background subtraction: three different ImageQuant automatic background subtraction methods were employed, and the results averaged for a best-estimate of expression differences.
Arystarkhova E., Haq I.U., Luebbert T., Mochel F., Saunders-Pullman R., Bressman S.B., Feschenko P., Salazar C., Cook J.F., Demarest S., Brashear A., Ozelius L.J, & Sweadner K.J. (2019). Factors in the disease severity of ATP1A3 mutations: impairment, misfolding, and allele competition. Neurobiology of disease, 132, 104577.
+ Open protocol
+ Expand
3

Western Blot Analysis of Protein Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols
The whole-cell extracts were prepared in lysis buffer with a protease inhibitor cocktail (Bimake), while the xenograft tissue extracts were prepared in 0.5% Nonidet P 40 Substitute solution (Biosharp) with cocktail protease inhibitor (Bimake). Each sample containing 30 μg of total protein was loaded onto 10% SDS-PAGE and electrotransferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skimmed milk in PBST at room temperature for 2 hours and incubated with primary antibodies at 4° C overnight. After the membranes were washed with PBST three times, they were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody at room temperature for 1 hour. Chemiluminescent detection was performed with WesternBright ECL (Advansta). The immunoblots were visualized using the FluorChem FC3 system (ProteinSimple). Antibodies: AR (SANTA, sc-7305), PSA (SANTA, sc-7316), acetylated α-tubulin (CST, 5335s), and α-tubulin (Proteintech, 66031-1-lg).
Zhou M., Zheng H., Li Y., Huang H., Min X., Dai S., Zhou W., Chen Z., Xu G, & Chen Y. (2021). Discovery of a novel AR/HDAC6 dual inhibitor for prostate cancer treatment. Aging (Albany NY), 13(5), 6982-6998.
+ Open protocol
+ Expand
4

Western Blot Protein Detection

Check if the same lab product or an alternative is used in the 5 most similar protocols
Briefly, protein bands separated on SDS-PAGE were transferred to a methanol-activated polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in 5% skim milk in PBST for 1 h. Subsequently, the membrane was incubated with 10 μg mL−1 primary antibody or Fab followed by incubation with goat-anti-human IgG-HRP (Thermo Fisher Scientific, cat. #31413, diluted 10,000×) or anti-his-HRP (Abcam, cat. #ab18184, diluted 1000×). Three washes for 5 min in PBST were conducted after each incubation step. WesternBright ECL (Advansta) was added for protein visualization.
Gu Y., Wong Y.H., Liew C.W., Chan C.E., Murali T.M., Yap J., Too C.T., Purushotorman K., Hamidinia M., El Sahili A., Goh A.T., Teo R.Z., Wood K.J., Hanson B.J., Gascoigne N.R., Lescar J., Vathsala A, & MacAry P.A. (2019). Defining the structural basis for human alloantibody binding to human leukocyte antigen allele HLA-A*11:01. Nature Communications, 10, 893.
+ Open protocol
+ Expand
5

Spike Protein Detection on LV Particles

Check if the same lab product or an alternative is used in the 5 most similar protocols
To evaluate spike presence on LV particles, pellets of concentrated preparations were resuspended and lysed in SDS loading buffer. Lysed virions were separated on 10% SDS polyacrylamide gel under reducing conditions and transferred to a nitrocellulose membrane with a Trans-Blot Turbo System (Bio-Rad, CA, USA). Filters were saturated for 1 h with 5% nonfat dry milk in TSBS (TBS with 0.1% Tween 20) and then incubated with polyclonal anti-spike (Sino Biological, Prodotti Gianni, Italy, Cat. 40590-T62; used at dilution 1/2000) or anti-Gag antibody (NIH Repository Reagents, USA, Cat. #4250; used at dilution 1/400), as primary antibodies, for 2 h at room temperature, followed by incubation for 1 h at room temperature with a goat anti-rabbit horse radish peroxidase (HRP)-conjugated IgG (Bio-Rad, Hercules, USA, Cat. 170-6515; dilution 1/3000). The immunocomplexes were visualized using the chemiluminescence ECL detection system (WesternBright ECL, Advansta).
Dispinseri S., Secchi M., Pirillo M.F., Tolazzi M., Borghi M., Brigatti C., De Angelis M.L., Baratella M., Bazzigaluppi E., Venturi G., Sironi F., Canitano A., Marzinotto I., Tresoldi C., Ciceri F., Piemonti L., Negri D., Cara A., Lampasona V, & Scarlatti G. (2021). Neutralizing antibody responses to SARS-CoV-2 in symptomatic COVID-19 is persistent and critical for survival. Nature Communications, 12, 2670.
+ Open protocol
+ Expand
6

Investigating Cell Signaling Pathways

Check if the same lab product or an alternative is used in the 5 most similar protocols
Proteins were extracted from cells and then used for western blot analysis. Drug treated cells were lysed with RIPA buffer containing a protease inhibitor cocktail and a phosphatase inhibitor cocktail (CWBIO, USA). Proteins were resolved by SDS-PAGE, transferred to PVDF membranes (Bio-Rad, USA), blocked with 5% skimmed milk powder. Then, the membranes were detected by incubation with 1:1000 dilutions of primary antibodies, washed, and incubated with Goat anti-rabbit-HRP antibodies and developed using WesternBright™ ECL (Advansta, USA). The following primary antibodies were used for western blot analysis: anti-p-IRE1α antibody, anti-IRE1α antibody, anti-CHOP antibody, anti-Cleaved caspase 3, anti-caspase 12, anti-Bcl2 Ser70, anti-p-p38 antibody, anti-p38 antibody (Cell Signaling Technology, USA), anti-β-catenin and anti-GAPDH (Proteintech, USA).
Wang Y., Wang C.Q., Sun P., Liu P.P., Yang H., Wang H.Y., Rao H.L., Li S., Jiang W.Q., Huang J.J, & Li Z.M. (2022). Phase II Study of Gemcitabine, Peg-Asparaginase, Dexamethasone and Methotrexate Regimen for Newly Diagnosed Extranodal Natural Killer/T-Cell Lymphoma, Nasal Type: Final Analysis With Long-Term Follow-Up and Rational Research for the Combination. Frontiers in Oncology, 12, 796738.
+ Open protocol
+ Expand
7

Collagen I Western Blot Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells were lysed using RIPA buffer containing 0.1% SDS, homogenized by passing lysate through a 20-gauge needle, placed on ice for 45 minutes, and centrifuged at 12000 rpm for 15 minutes at 4 °C. Protein concentrations of the supernatant were determined using a BCA Assay (Thermo Scientific, Rockford, IL USA) according to manufacturer recommendations. 30 μg of protein/sample were separated by gel electrophoresis in a 4–12% Bis-Tris Gel (Life Technologies, CA) and transferred to Immobilon membranes (Millipore, Billerica, MA). Membranes were blocked with a solution of 2.5% albumin and 2.5% non-fat milk in TBST (Cell Signaling Technology, Beverly, MA) and subsequently incubated with rabbit anti-collagen I antibody (1:2500) (Abcam, Cambridge, MA) overnight, followed by a 1-hour incubation with donkey anti-rabbit secondary antibody (1:5000) (Abcam). Blots were developed with WesternBright ECL (Advansta, Menlo Park, CA) and imaged on Blue Ultra AutoRad Film (BioExpress, Kaysville, UT).
Muir A.B., Dods K., Noah Y., Toltzis S., Chandramouleeswaran P.M., Lee A., Benitez A., Bedenbaugh A., Falk G.W., Wells R.G., Nakagawa H, & Wang M.L. (2014). Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition. Experimental cell research, 330(1), 102-110.
+ Open protocol
+ Expand
8

Western Blot Analysis of Protein Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cultured cells were lysed in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.8% NP-40, 0.2% Triton X-100, 3% glycerol). Cell lysates were subjected to polyacrylamide gel electrophoresis (PAGE), and proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. Membranes were soaked in 5% nonfat milk or 5% BSA solution for one hour to block nonspecific binding of proteins, and then incubated with primary antibodies overnight at 4 °C. On the following day, membranes were incubated with secondary antibodies for 2 h at room temperature, and WesternBright ECL (Advansta, Menlo Park, CA) was used with the luminescent image analyzer (Jun Yi Dong Fang, Beijing, China) to capture images. Uncropped scans can be found in Figure S1.
Antibodies used in western blotting were: KLF5 (1:1000, 21017-1-AP, Proteintech), AR (1:1000, 5153S, Cell Signaling), PSA (1:3000, 10679-1-AP, Proteintech), MYC (1:1000, 9402, Cell Signaling), cyclin D1 (1:10000, ab134175, Abcam), FLAG (1:3000, SAB4200071, Sigma), HA (1:3000, 3724S, Cell Signaling), GAPDH (1:3000, 60004-1-Ig, Proteintech).
Li J., Zhang B., Liu M., Fu X., Ci X., A J., Fu C., Dong G., Wu R., Zhang Z., Fu L, & Dong J.T. (2020). KLF5 Is Crucial for Androgen-AR Signaling to Transactivate Genes and Promote Cell Proliferation in Prostate Cancer Cells. Cancers, 12(3), 748.
+ Open protocol
+ Expand
9

Western Blot Protein Extraction and Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Total cell protein was extracted using protein lysis buffer (20 mmol/L Tris pH 7.4, 150 mmol/L NaCl, 1% Igepal CA-630, 5 mmol/L Ethylenediaminetetraacetic acid [EDTA]) containing protease and phosphatase inhibitor cocktails and phenylmethyl sulfonyl fluoride (Sigma-Aldrich). Bicinchoninic acid protein analysis (Thermo Fisher Scientific) was used to determine equal concentrations of protein for each sample lysate. Samples were then boiled for 5 minutes at 95°C in loading dye containing SDS, separated by 1.5 mm SDS-PAGE gels, and then transferred onto nitrocellulose membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were incubated overnight in primary antibody, washed, and incubated with horseradish peroxidase–conjugated anti-rabbit Ig (RRID:AB_2099233) or anti-mouse Ig (RRID:AB_330924; Cell Signaling Technology) for 1 hour. Membranes were then visualized on the ChemiDoc Imaging System (Bio-Rad) following development with WesternBright ECL or Sirius (Advansta) according to the manufacturer's instructions. Primary antibodies used are listed in Supplementary Table S2.
Eiken A.P., Smith A.L., Skupa S.A., Schmitz E., Rana S., Singh S., Kumar S., Mallareddy J.R., de Cubas A.A., Krishna A., Kalluchi A., Rowley M.J., D'Angelo C.R., Lunning M.A., Bociek R.G., Vose J.M., Natarajan A, & El-Gamal D. (2024). Novel Spirocyclic Dimer, SpiD3, Targets Chronic Lymphocytic Leukemia Survival Pathways with Potent Preclinical Effects. Cancer Research Communications, 4(5), 1328-1343.
+ Open protocol
+ Expand
10

Western Blot Analysis of Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells cultured in 6-well plates were rinsed with PBS buffer, harvested into centrifuge tubes then resuspended in western blot or IP lysis buffer with the proteinase inhibitor PMSF (Beyotime Biotechnology, #ST506) and incubated on ice for 45 min. Concentration of proteins was determined using a BCA Protein assay Kit (Beyotime Biotechnology, #P0012S). Same amounts of proteins were loaded and separated by 12% SDS-PAGE, and then transferred onto 0.22-μm PVDF membranes (Millipore, Billerica, MA, United States). Membranes were blocked with 5% skim milk at room temperature for 2 h and then incubated with 2% blocking solution containing the primary antibodies at the dilution suggested by the manufacturer at 4°C overnight. The membranes were then washed three times with TBST and incubated with the secondary HRP-conjugated antibodies diluted at 1: 5000 in 2% blocking solution for 2 h at room temperature. After washed another three times, the immunoreactive bands were detected by using chemiluminescent detection reagent WesternBright ECL (Advansta, United States, #R-03026-D100) and analyzed by Tanon-410 automatic gel imaging system (Shanghai Tianneng, China).
Lin J., Wang C., Zhang L., Wang T., Zhang J., Liang W., Li C., Qian G., Ouyang Y., Guo K, & Zhang Y. (2017). Rab5 Enhances Classical Swine Fever Virus Proliferation and Interacts with Viral NS4B Protein to Facilitate Formation of NS4B Related Complex. Frontiers in Microbiology, 8, 1468.
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!