Western lightning ecl pro kit

Manufactured by PerkinElmer

Sourced in United States

The Western Lightning ECL Pro kit is a chemiluminescent detection system designed for the analysis of proteins in Western blotting experiments. It provides a sensitive and reliable method for the detection of target proteins.

Automatically generated - may contain errors

Lab products found in correlation

Bcl 2, by Abcam (1 mentions) Pierce 660 nm protein assay reagent, by Thermo Fisher Scientific (1 mentions) Bcl xl, by Cell Signaling Technology (1 mentions) Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions) Gapdh, by Abcam (1 mentions) Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Pvdf membrane, by Pall Corporation (1 mentions) Subcellular protein fraction kit, by Thermo Fisher Scientific (1 mentions) Block ace powder, by Sumitomo Pharma (1 mentions) Qubit 3 fluorometer, by Thermo Fisher Scientific (1 mentions) Anti rabbit hrpantibody, by Promega (1 mentions) Trans blot turbo transfer system, by Bio-Rad (1 mentions) Anti flag primary antibody, by Merck Group (1 mentions) Mini trans blot cell, by Bio-Rad (1 mentions) Horseradish peroxidase hrp, by Merck Group (1 mentions) Mini protean tetra cell, by Bio-Rad (1 mentions) Snai2, by Cell Signaling Technology (1 mentions) Twist, by Santa Cruz Biotechnology (1 mentions)

9 protocols using western lightning ecl pro kit

Purification and Analysis of FtsZ Derivatives

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

FtsZ derivatives were purified by precipitation with ammonium sulfate as described (Haeusser et al., 2014 (link)). After resuspension in Storage Buffer (50 mM Tris pH 7.5, 250 mM KCl, 10 mM MgCl₂, 1 mM EDTA, and 10% glycerol), GDP (0.05 mM) was added followed by freezing in liquid nitrogen and storage at −80°C.
His₆-tagged FtsA and FtsA*-like variants were purified using Talon metal affinity resin as described (Krupka et al., 2017 (link)). The purified proteins (Fig. S6) were resuspended in Storage Buffer supplemented with 0.05 mM ADP, frozen in liquid nitrogen and stored at −80°C. The CB-X protein estimation assay (G-Biosciences) was used to determine protein concentration.
Cell extracts were processed for immunoblotting as described (Haeusser et al., 2014 (link)), using anti-FLAG (1:2000), affinity purified polyclonal anti-FtsZ (1:5000), and primary and goat anti-rabbit secondary antibodies (1:2000, Sigma) conjugated to horseradish peroxidase. Western Lightning ECL Pro kit (PerkinElmer) was used to detect chemiluminescence. Protein band intensities were measured and compared using ImageJ (Schneider et al., 2012 (link)).

Schoenemann K.M., Krupka M., Rowlett V.W., Distelhorst S.L., Hu B, & Margolin W. (2018). Gain-of-function variants of FtsA form diverse oligomeric structures on lipids and enhance FtsZ protofilament bundling. Molecular microbiology, 109(5), 676-693.

+ Open protocol

+ Expand

Molecular Analysis of Apoptotic Signaling Pathways

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were washed twice in PBS and lysed using M-PER mammalian protein extraction reagent (Thermoscientific) according to the manufacturer’s recommendations. Protein concentrations were quantified using the Pierce 660-nm protein assay reagent (Thermoscientific) according to the manufacturer’s suggested protocol. Whole-cell lysates (20 μg) were resolved by SDS-polyacrylamide gel electrophoresis on 10%–15% gels, and transferred to polyvinylidene difluoride membranes (Immobilon; Amersham, Arlington Heights, IL). Membranes were then probed sequentially with antibodies against Bcl-2 (1∶1,000; Epitomics, Burlingame, CA), Bcl-xL (1∶1,000; Cell Signaling Technology), Bax (1∶1,000; Cell Signaling Technology), cleaved caspase-3 (1∶1000; Cell Signaling Technology), and GAPDH (1∶10,000; Abcam, Cambridge, UK). The blots were developed using an enhanced chemiluminescence substrate (Western Lightning ECL Pro) kit (PerkinElmer, San Jose, CA).

Koyama N., Nishida Y., Ishii T., Yoshida T., Furukawa Y, & Narahara H. (2014). Telmisartan Induces Growth Inhibition, DNA Double-Strand Breaks and Apoptosis in Human Endometrial Cancer Cells. PLoS ONE, 9(3), e93050.

+ Open protocol

+ Expand

Western Blot Analysis of Cell Fractions

Check if the same lab product or an alternative is used in the 5 most similar protocols

Proteins extracted from the immunoprecipitants of whole cells, the microsomal fraction, the cytosolic fraction, or nuclear fraction were resolved on 10 or 15% SDS-polyacrylamide gels and electroblotted onto PVDF membranes as described by Yabu et al.^{16 (link), 60 (link)} Anti-FLAG monoclonal, anti-V5 monoclonal, anti-nSMase1 polyclonal,^{16 (link)} anti-nSMase1/SMPD2 monoclonal, anti-phospho-nSMase1 polyclonal, anti-actin monoclonal, anti-pan-cadherin polyclonal, anti-aldolase polyclonal, anti-tubulin monoclonal, anti-JNK polyclonal, rabbit anti-phospho-JNK (Thr183/Tyr185) monoclonal, mouse anti-phospho-JNK (Thr183/Tyr185) monoclonal, anti-phospho-MKK4 (Ser257/Thr261) polyclonal, anti-phospho-MKK7 (Ser271/Thr275) polyclonal, anti-phospho-c-Jun (Ser73) polyclonal, anti-SEK1/MKK4 (5C10) monoclonal, anti-MKK7 polyclonal, and anti-histone H2A.Z polyclonal primary antibodies were used. Following reactions with the appropriate secondary antibodies, the resulting signals were detected by chemiluminescence using a Western Lightning ECL Pro kit (PerkinElmer Inc., Waltham, MA, USA) according to the manufacturer's instructions.

Yabu T., Shiba H., Shibasaki Y., Nakanishi T., Imamura S., Touhata K, & Yamashita M. (2014). Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling. Cell Death and Differentiation, 22(2), 258-273.

+ Open protocol

+ Expand

Western Blot Analysis of Protein Lysates

Check if the same lab product or an alternative is used in the 5 most similar protocols

KERTr cells (2 × 10⁶ cells/well) were lysed in protein lysis buffer, sonicated, and centrifuged at 13,000g for 10 minutes at 4°C to remove cell debris. Protein concentration was determined using the micro-BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of protein (12 μg/sample) were separated using SDS-PAGE and transferred to PVDF membranes (PALL Corporation). Membranes were blocked with Tris-buffered saline and Tween 20 containing 5% BSA. The primary antibodies for immunoblotting are listed in Supplemental Table 3. The primary antibodies were detected with peroxidase-conjugated anti–rabbit IgG or anti–mouse IgG, followed by detection with Western Lightning ECL Pro kit (PerkinElmer) according to the manufacturer’s recommendations (PerkinElmer, NEL120E001EA).

Luo C.H., Lai A.C., Tsai C.C., Chen W.Y., Chang Y.S., Chung E.J, & Chang Y.J. (2024). Staphylococcus aureus exacerbates dermal IL-33/ILC2 axis activation through evoking RIPK3/MLKL-mediated necroptosis of dry skin. JCI Insight, 9(6), e166821.

+ Open protocol

+ Expand

Nuclear Protein Extraction and Western Blot

Check if the same lab product or an alternative is used in the 5 most similar protocols

Nuclear proteins from SE tissues of E16.5 were extracted using a Subcellular Protein Fraction Kit (ThermoFisher, cat. No.78840). Nucleoprotein extracts were quantified using a Qubit 3 Fluorometer (ThermoFisher), after which 40 μg of each extract was loaded onto a 10% bis-acrylamide gel and subjected to SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred to a polyvinylidene fluoride membrane using a Trans-Blot turbo transfer system (BioRad). The membrane was blocked using Block Ace Powder (DS Pharma Biomedical) for 2 h at RT. After primary incubation using blocking solution with anti-GRHL3 antibody (1:1000 antigen aa195-211) overnight at 4 °C, a second incubation was performed using blocking solution with anti-rabbit HRP antibody (Promega). Finally, the signal was revealed using a Western Lightning ECL Pro Kit (Perkin Elmer, Waltham, MA, USA; cat. No. NEL120001EA).

Kimura-Yoshida C., Mochida K., Nakaya M.A., Mizutani T, & Matsuo I. (2018). Cytoplasmic localization of GRHL3 upon epidermal differentiation triggers cell shape change for epithelial morphogenesis. Nature Communications, 9, 4059.

+ Open protocol

+ Expand

SDS-PAGE and Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Crude extracts, eluates, and purified proteins were resuspended in 5X SDS loading buffer (0.2 M Tris-HCl (pH 6.8), 25% glycerol, 0.5 M DTT, 10% SDS, 0.05% bromophenol blue), boiled for 10 min, and separated by SDS-PAGE in a Mini PROTEAN Tetra Cell (Bio-Rad) using 12.5% polyacrylamide gels. Gels were stained with Coomassie protein stain for at least one hour followed by incubation in destaining solution.
For Western Blot analysis proteins were transferred to nitrocellulose membranes using a Bio-Rad Mini Trans-Blot Cell. Mouse monoclonal anti-FLAG primary antibodies were purchased from Sigma-Aldrich. Polyclonal anti-ZipA serum was affinity purified according to Levin, 2002. Primary antibody dilutions of 1:5,000 or 1:10,000 were used. Anti-mouse and anti-rabbit secondary antibodies conjugated to horseradish peroxidase (HRP) (Sigma-Aldrich) were used at a 1:10,000 or 1:5000 dilutions. Western Lightning ECL Pro kit (PerkinElmer) was used to detect chemiluminescence.

Herricks J.R., Nguyen D, & Margolin W. (2014). A thermosensitive defect in the ATP binding pocket of FtsA can be suppressed by allosteric changes in the dimer interface. Molecular microbiology, 94(3), 713-727.

+ Open protocol

+ Expand

Protein Expression Analysis in Palatal Tissue

Check if the same lab product or an alternative is used in the 5 most similar protocols

Lysates of the palate shelves were extracted using 2X sample buffer (100 mM Tris pH6.8, 0.1 M MgCl₂.6H₂O, 2% SDS, 5% glycerol, 2.5% beta-mercaptoethanol, 2.5% bromophenol blue), and heated at 95 ^oC for 5 minutes. The lysates were then centrifuged at 4 ^oC, 14000 rpm for 1 hr, and the supernatant collected. Total proteins were separated by 10% SDS-PAGE. Western blotting analysis was performed by incubating with antibodies against IRF6 (Genetex), E-cadherin (Cell signaling technology), ZO-1 (Invitrogen), Plakophilin (Plakophilin-1; Abcam), TWIST (Santa Cruz biotechnology), SNAI2 (Cell signaling technology), or GAPDH (Merck Millipore) overnight at 4^oC. Subsequently, the membranes were incubated with anti-mouse IgG-HRP or anti-rabbit IgG-HRP secondary antibodies (GE Healthcare) at room temperature for 1 hr, and the signals detected using a Western Lightning ECL Pro kit (PerkinElmer).

Ke C.Y., Xiao W.L., Chen C.M., Lo L.J, & Wong F.H. (2015). IRF6 is the mediator of TGFβ3 during regulation of the epithelial mesenchymal transition and palatal fusion. Scientific Reports, 5, 12791.

+ Open protocol

+ Expand

Western Blot Analysis of Bacterial Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell extracts were prepared, and proteins were separated by SDS–polyacrylamide gel electrophoresis and transferred as previously described61 (link). Cellular levels of FtsZ or ZapA were measured on immunoblots by affinity purified rabbit anti-E. coli FtsZ (lab collection; 1:5,000 dilution)70 (link) or rabbit anti-E. coli ZapA antiserum (lab collection; 1:2,000 dilution). Rabbit anti-FLAG antibody (Sigma-Aldrich F7425) was used to measure FLAG-tagged FtsA levels (1:4,000 dilution). Goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (Sigma-Aldrich AQ132P) were used at 1:10,000 dilution. A Western Lightning ECL Pro Kit (Perkin-Elmer) was used for horseradish peroxidase detection and blots were developed using an ImageQuant LAS 4000 mini-image analyser.

Krupka M., Rowlett V.W., Morado D., Vitrac H., Schoenemann K., Liu J, & Margolin W. (2017). Escherichia coli FtsA forms lipid-bound minirings that antagonize lateral interactions between FtsZ protofilaments. Nature Communications, 8, 15957.

+ Open protocol

+ Expand

Protein Profiling of PDX Tumor Lysates

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

For each PDX model, four tumors were randomly selected and tumor lysates were prepared from snap frozen tumor fragments as described previously [38 (link)]. The lysates were then loaded on NuPAGE™ 4%–12% Bis-Tris Gels (ThermoFisher Scientific) and electrophoresed using NuPage™ MOPS SDS Running Buffer (ThermoFisher Scientific) at 150 V for 2 h. Four different tumor lysates were used per treatment group. Samples were then blotted on polyvinylidene fluoride membranes (Bio-Rad) in a semi-dry transfer at 25 V for 30 min, using a transfer buffer with 20% methanol, 25 mM tromethamine and 190 mM glycine. The following primary antibodies were used: KIT (A450229-2, Agilent), phospho-KIT Tyr719, phospho-KIT Tyr703, AKT, phospho-AKT Ser743, p44/42 mitogen-activated protein kinase (MAPK), phospho-p44/42 MAPK Thr202/Tyr204, eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), phospho-4E-BP1 Ser65, ribosomal protein S6, phospho-S6 Ser240/244 and α-tubulin (catalogue numbers: 3391, 3073, 7292, 7291, 9102, 4370, 9644, 9456, 2217, 5364 and 2144, respectively; all from Cell Signaling Technology). Horseradish peroxidase conjugated secondary polyclonal goat antirabbit immunoglobulins (P044801, Agilent) were added and specific bands were visualized using Western Lightning™ ECL Pro kit (Perkin Elmer). Chemiluminescence was captured using the FUJI-LAS mini 3000 system (Fujifilm).

Wang Y., Wozniak A., Wellens J., Gebreyohannes Y.K., Guillén M.J., Avilés P.M., Debiec-Rychter M., Sciot R, & Schöffski P. (2020). Plocabulin, a novel tubulin inhibitor, has potent antitumor activity in patient-derived xenograft models of gastrointestinal stromal tumors. Translational Oncology, 13(11), 100832.

+ 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?

Revolutionizing how scientists
search and build protocols!