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Western lightening plus

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Western Lightening Plus is a chemiluminescent detection reagent used for the visualization of target proteins in Western blot analysis. It is designed to provide high-intensity, long-lasting signal for sensitive detection of low-abundance proteins.

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Lab products found in correlation

Streptavidin hrp, by Vector Laboratories (2 mentions) Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions) Concanavalin a (cona), by Vector Laboratories (1 mentions) Pvdf membrane, by PerkinElmer (1 mentions) Jla20, by Developmental Studies Hybridoma Bank (1 mentions)

Close spelling variants of this product

Western Lightening Plus ECL Western Lightening™ Plus-ECL (Enhanced Chemiluminescence) Substrate assay kit Western Lightening Chemiluminescence Reagent Plus Kit Western Lightening® Chemiluminescence Reagent Plus Western Lightening Plus-ECL substrate Western Lightening Plus – Enhanced Chemiluminescence Substrate Western Lightening Plus -ECL Kit Western Lightening

4 protocols using western lightening plus

1

Knockdown of STC1 in THP-1 macrophages

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On-Target human STC1 siRNA (Dharmacon) was used to knock down the STC1 transcript. Non-targeting siRNA: UGGUUUACAUGUCGACUAA, UGGUUUACAUGUUGUGUGA, UGGUUUAC AUGUUUUCUGA, UGGUUUACAUGUUUUCCUA, and human STC1 siRNA: AAACGCACAUCCCAUGAGA, GGGAAAAGCAUUCGUCAAA, GUACAGCG CUGCUAAAUUU, CAACAGAUACUAUAACAGA were used in this study. Briefly, 1 × 105 ThP-1 cells were seeded in 24-well plates, then transfected with 50 nM siRNA using 0.5 μl Lipofectamine 2000 transfection reagent (Invitrogen; Life Technologies) in antibiotic-free complete medium. After 6 h incubation, the siRNA transfected cells were treated with 5 nM PMA to induce M0 macrophage differentiation. In some experiments, the siRNA transfected M0 macrophages were further stimulated to obtain M1 or M2 polarized cells. The decrease in the expression levels of STC1 mRNA and protein were validated by real-time PCR and western blotting. Western blot was conducted using a rabbit antibody to STC1 (Sigma), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody. Specific bands were visualized with chemiluminescent reagent (Western-lightening Plus, PerkinElmer Life Sciences). Blots were then washed in PBS and re-probed with rabbit anti-actin serum (Sigma).
Leung C.C, & Wong C.K. (2020). Characterization of stanniocalcin-1 expression in macrophage differentiation. Translational Oncology, 14(1), 100881.
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2

Lectin Blot Analysis of Glycosylation

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The cells were lysed in RIPA buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitor. The lysate was cleared by centrifugation at 10 000× g for 10 min. Protein concentrations were determined by BCA assay (Pierce, Rockford, IL). Twenty μg of proteins were separated on 4–12% NuPage Bis Tris gels and transferred to PVDF membrane at 25 V for 1.5 h. Membranes were blocked overnight in 3% BSA/TBST buffer before lectin blot detection using a 1:5000 dilution of the following biotinylated lectins: (Sambucus nigra (SNL), (SNL), Aleuria aurantia (AAL), and Datura stramonium (DSL), Concanavalin A (ConA) (Vector Labs). Bound lectin was detected using a 1:10,000 dilution of streptavidin–HRP (Vector Labs) before washing and detection using Western Lightening Plus (Perkin Elmer).
Murali P., Johnson B.P., Lu Z., Climer L., Scott D.A., Foulquier F., Oprea-Ilies G., Lupashin V., Drake R.R, & Abbott K.L. (2020). Novel role for the Golgi membrane protein TMEM165 in control of migration and invasion for breast carcinoma. Oncotarget, 11(28), 2747-2762.
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3

Slot Blot Analysis of Alpha Toxin Binding

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Plasma (5 μl) was mixed with 5 μl Laemelli buffer. Samples were heated to 95°C for 2 minutes to denature the proteins and cooled to room temperature before application to the membrane. Nitrocellulose Protran BA85 membrane was immobilized and clamped securely using a Schleicher and Schuell Minifold I Slot Blot System. The samples were added to each slot before the vacuum was applied for 1 minute. Each well was washed using 1X PBS (200 μl) and again the vacuum was applied for 1 minute before removal of the membrane from the apparatus. The membrane was blocked in 5% milk/1X TBST (Blotto Solution) overnight at 4°C. The blot was incubated with biotin labeled alpha toxin (2 μg/ml) expressed, purified, and labeled as described previously [4 (link)]. Bound toxin was detected using a 1:5,000 dilution of streptavidin-HRP (Vector Labs, Burlingame, CA) before washing and detection using Western Lightening Plus (Perkin Elmer). Slot blots were then stripped using 0.1 M glycine pH 2.9 overnight, blocked again, and detected using anti-alpha 1 acid glycoprotein antibody (Sigma) to normalize for total protein content. Intensity of alpha toxin binding was determined using ImageJ analysis and was normalized to total protein band density.
Dolezal S., Hester S., Kirby P.S., Nairn A., Pierce M, & Abbott K.L. (2014). Elevated levels of glycosylphosphatidylinositol (GPI) anchored proteins in plasma from human cancers detected by C. septicum alpha toxin. Cancer biomarkers : section A of Disease markers, 14(1), 55-62.
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4

Western Blot Analysis of Protein Lysates

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Protein lysates were subjected to electrophoresis in 8% polyacrylamide gels. Lysates contained 20-40 mg of total protein in a radioimmunoprecipitation assay buffer mixed with 5! SDS loading buffer (250 mM Tris-HCl at pH 6.8, 10% SDS, 50% glycerol, 5% b-mercaptoethanol, 62.5 mM EDTA, and 0.1% bromophenol blue). Postelectrophoresis, the gels were blotted onto PVDF membranes (PerkinElmer Life Sciences). Western blotting was conducted using rabbit antibodies against STC1 (Origene, Rockville, MD, USA) and AP (GenHunter) as well as mouse antibodies against V5 (Invitrogen) and His (GE Healthcare Life Science), and then the gels were incubated with HRP-conjugated goat anti-rabbit or antimouse antibody (1:4000; Bio-Rad). Specific bands were visualized using a chemiluminescent reagent (Western-Lightening Plus, PerkinElmer Life Sciences). For some experiments, the blots were then washed in PBST and re-probed with mouse monoclonal anti-fish actin antibody (1:100; JLA20, Developmental Studies Hybridoma Bank).
Gu J., Law A.Y., Yeung B.H, & Wong C.K. (2015). Characterization of stanniocalcin 1 binding and signaling in gill cells of Japanese eels. Journal of molecular endocrinology, 54(3).
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