Western lightning oxidizing and luminol reagents

Manufactured by PerkinElmer

Sourced in United States

Western Lightning oxidizing and luminol reagents are chemiluminescent detection reagents used in Western blotting techniques to visualize and quantify proteins. The oxidizing reagent enhances the chemiluminescent signal, while the luminol reagent produces the light-emitting reaction. These reagents are designed to enable sensitive and reliable protein detection.

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Lab products found in correlation

Lb942 tristar luminometer, by Berthold Technologies (7 mentions) Maxisorp, by Thermo Fisher Scientific (6 mentions) Tristar lb 941 luminometer, by Berthold Technologies (3 mentions) Hrp conjugated anti igg secondary abs, by Thermo Fisher Scientific (2 mentions)

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Western Lightning (69 citations) Western Lightning reagent (17 citations) Luminol (3 citations)

10 protocols using western lightning oxidizing and luminol reagents

Quantifying Anti-gp120 Antibody Binding

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The gp120 inner domain stabilized ID2 (Tolbert et al., 2016 (link)) (0.1μg/ml) was diluted in PBS. Bovine serum albumin (BSA) (0.1μg/ml) was used a negative control. Proteins were adsorbed to plates (MaxiSorp; Nunc) overnight at 4°C. Coated wells were subsequently blocked with blocking buffer (tris-buffered saline (TBS) containing 0.1% Tween-20 and 2% (w/v) BSA for 1:30 hour at room temperature. HIV+ sera (1:1000 dilution) were diluted in blocking buffer and added to the coated wells for 1:30 hour at room temperature. Wells were washed four times with washing buffer (TBS containing 0.1% Tween-20). Horseradish peroxidase-conjugated (HRP) antibody specific of human IgG (Pierce) was added for 1 hour at room temperature, followed by four washes. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences). Light emission was measured with the LB 941 TriStar luminometer (Berthold Technologies).

Alsahafi N., Bakouche N., Kazemi M., Richard J., Ding S., Bhattacharyya S., Das D., Anand S.P., Prévost J., Tolbert W.D., Lu H., Medjahed H., Gendron-Lepage G., Delgado G.G., Kirk S., Melillo B., Mothes W., Sodroski J., Smith AB I.I.I., Kaufmann D.E., Wu X., Pazgier M., Rouiller I., Finzi A, & Munro J.B. (2019). An Asymmetric Opening of HIV-1 Envelope Mediates Antibody-Dependent Cellular Cytotoxicity. Cell host & microbe, 25(4), 578-587.e5.

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SARS-CoV-2 RBD Antibody ELISA Assay

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The SARS-CoV-2 RBD ELISA was used as described previously (6 (link), 7 (link)). Briefly, recombinant SARS-CoV-2 S RBD protein (2.5 μg/mL), or bovine serum albumin (BSA) (2.5 μg/mL) as a negative control, was prepared in phosphate-buffered saline (PBS) and adsorbed to plates (MaxiSorp Nunc) overnight at 4°C. Coated wells were subsequently blocked with blocking buffer (Tris-buffered saline [TBS] containing 0.1% Tween 20 and 2% BSA) for 1 h at room temperature. Wells were then washed four times with washing buffer (TBS containing 0.1% Tween 20). CR3022 monoclonal antibody (MAb) (50 ng/mL) or a 1:250 dilution of plasma samples was prepared in a diluted solution of blocking buffer (0.1% BSA) and incubated with the RBD-coated wells for 90 min at room temperature. Plates were washed four times with washing buffer followed by incubation with secondary antibodies (Abs; diluted in a diluted solution of blocking buffer [0.4% BSA]) for 1 h at room temperature, followed by four washes. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences). Light emission was measured with a LB942 TriStar luminometer (Berthold Technologies). Signal obtained with BSA was subtracted for each plasma and was then normalized to the signal obtained with CR3022 present in each plate.

Valcourt E.J., Manguiat K., Robinson A., Lin Y.C., Abe K.T., Mubareka S., Shigayeva A., Zhong Z., Girardin R.C., DuPuis A., Payne A., McDonough K., Wang Z., Gasser R., Laumaea A., Benlarbi M., Richard J., Prévost J., Anand S.P., Dimitrova K., Phillipson C., Evans D.H., McGeer A., Gingras A.C., Liang C., Petric M., Sekirov I., Morshed M., Finzi A., Drebot M, & Wood H. (2021). Evaluating Humoral Immunity against SARS-CoV-2: Validation of a Plaque-Reduction Neutralization Test and a Multilaboratory Comparison of Conventional and Surrogate Neutralization Assays. Microbiology Spectrum, 9(3), e00886-21.

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Quantification of Soluble gp120 by ELISA

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The concentration of soluble gp120 in the supernatant of gp160-expressing cells was determined by an anti-gp120 sandwich ELISA. A combination of nnAbs 17b and A32 was prepared in PBS and adsorbed to plates (MaxiSorp; Nunc) overnight at 4 °C. The coated wells were subsequently blocked with a blocking buffer (Tris-buffered saline (TBS) containing 0.1% Tween 20 and 2% BSA) for 90 min at room temperature. The wells were then washed four times with a washing buffer (Tris-buffered saline (TBS) containing 0.1% Tween 20). Supernatants from gp160-expressing cells were incubated with the nnAbs-coated wells for 120 min at room temperature. The plates were washed four times with a washing buffer, followed by incubation with HRP-conjugated C11 nnAbs (3 mg/mL) for 90 min at room temperature. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences, Waltham, MA, USA). Light emission was measured with an LB942 Tri-Star luminometer (Berthold Technologies, Bad Wildbad, Germany). A standard curve using known concentrations of purified recombinant gp120YU2 was used to quantify the precise concentration of soluble gp120 in each supernatant.

Boutin M., Medjahed H., Nayrac M., Lotke R., Gendron-Lepage G., Bourassa C., Sauter D., Richard J, & Finzi A. (2023). Temsavir Modulates HIV-1 Envelope Conformation by Decreasing Its Proteolytic Cleavage. Viruses, 15(5), 1189.

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SARS-CoV-2 Spike Protein ELISA Assay

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The SARS-CoV-2 Spike ELISA assay used was recently described (Beaudoin-Bussieres et al., 2020 (link); Prevost et al., 2020 (link)). Briefly, recombinant SARS-CoV-2 S-6P and RBD proteins (2.5 μg/ml), or bovine serum albumin (BSA) (2.5 μg/ml) as a negative control, were prepared in PBS and were adsorbed to plates (MaxiSorp; Nunc) overnight at 4 °C. Coated wells were subsequently blocked with blocking buffer (Tris-buffered saline [TBS] containing 0.1% Tween20 and 2% BSA) for 1 hour at room temperature. Wells were then washed four times with washing buffer (TBS containing 0.1% Tween20). CV3-1, CV3-25 and CR3022 mAbs (50 ng/ml) were prepared in a diluted solution of blocking buffer (0.1 % BSA) and incubated with the RBD-coated wells for 90 minutes at room temperature. Plates were washed four times with washing buffer followed by incubation with HRP-conjugated anti-IgG secondary Abs (Invitrogen) (diluted in a diluted solution of blocking buffer [0.4% BSA]) for 1 hour at room temperature, followed by four washes. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences). Light emission was measured with a LB941 TriStar luminometer (Berthold Technologies). Signal obtained with BSA was subtracted for each plasma and was then normalized to the signal obtained with CR3022 mAb present in each plate.

Ullah I., Prévost J., Ladinsky M.S., Stone H., Lu M., Anand S.P., Beaudoin-Bussières G., Symmes K., Benlarbi M., Ding S., Gasser R., Fink C., Chen Y., Tauzin A., Goyette G., Bourassa C., Medjahed H., Mack M., Chung K., Wilen C.B., Dekaban G.A., Dikeakos J.D., Bruce E.A., Kaufmann D.E., Stamatatos L., McGuire A.T., Richard J., Pazgier M., Bjorkman P.J., Mothes W., Finzi A., Kumar P, & Uchil P.D. (2021). Live imaging of SARS-CoV-2 infection in mice reveals that neutralizing antibodies require Fc function for optimal efficacy. Immunity, 54(9), 2143-2158.e15.

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SARS-CoV-2 Spike Protein ELISA Assay

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The SARS-CoV-2 Spike ELISA assay used was recently described (Beaudoin-Bussières et al., 2020 (link); Prévost et al., 2020 (link)). Briefly, recombinant SARS-CoV-2 S-6P and RBD proteins (2.5 μg/mL), or bovine serum albumin (BSA) (2.5 μg/mL) as a negative control, were prepared in PBS and were adsorbed to plates (MaxiSorp; Nunc) overnight at 4°C. Coated wells were subsequently blocked with blocking buffer (Tris-buffered saline [TBS] containing 0.1% Tween20 and 2% BSA) for 1 h at room temperature. Wells were then washed four times with washing buffer (TBS containing 0.1% Tween20). CV3-1, CV3-25 and CR3022 mAbs (50 ng/mL) were prepared in a diluted solution of blocking buffer (0.1% BSA) and incubated with the RBD-coated wells for 90 min at room temperature. Plates were washed four times with washing buffer followed by incubation with HRP-conjugated anti-IgG secondary Abs (Invitrogen) (diluted in a diluted solution of blocking buffer [0.4% BSA]) for 1 h at room temperature, followed by four washes. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences). Light emission was measured with a LB941 TriStar luminometer (Berthold Technologies). Signal obtained with BSA was subtracted for each plasma and was then normalized to the signal obtained with CR3022 mAb present in each plate.

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SARS-CoV-2 RBD-specific IgG ELISA Assay

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The SARS-CoV-2 RBD ELISA assay was used to measure the level of RBD-specific IgG, as previously described (Beaudoin-Bussieres et al., 2020 ; Prevost et al., 2020 ). Briefly, recombinant SARS-CoV-2 RBD protein was prepared in PBS (2.5 μg/ml) and adsorbed to plates overnight at 4°C. Coated wells were subsequently blocked with blocking buffer then washed. CR3022 monoclonal Ab (50ng/ml) at 1/250, 1/500, 1/1250, 1/2500, 1/5000, 1/10000, 1/20000 dilutions of plasma from SARS-CoV-2-naive or previously infected donors were prepared in a diluted solution of blocking buffer and incubated with the RBD-coated wells. Plates were washed followed by incubation with the respective secondary Abs. Area Under the Cure (AUC) was calculated by using GraphPad. To calculate the RBD-avidity index, we performed a stringent ELISA where the plate was washed with washing buffer supplemented 8M urea. The binding of CR3022 IgG and plasma was quantified with HRP-conjugated antibodies specific for the Fc region of human IgG. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences). Light emission was measured with a LB942 TriStar luminometer (Berthold Technologies).

Nayrac M., Dubé M., Sannier G., Nicolas A., Marchitto L., Tastet O., Tauzin A., Brassard N., Beaudoin-Bussières G., Vézina D., Gong S.Y., Benlarbi M., Gasser R., Laumaea A., Bourassa C., Gendron-Lepage G., Medjahed H., Goyette G., Ortega-Delgado G.G., Laporte M., Niessl J., Gokool L., Morrisseau C., Arlotto P., Richard J., Tremblay C., Martel-Laferrière V., Finzi A, & Kaufmann D.E. (2021). Temporal associations of B and T cell immunity with robust vaccine responsiveness in a 16-week interval BNT162b2 regimen. bioRxiv.

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SARS-CoV-2 Receptor Binding Domain ELISA

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The SARS-CoV-2 RBD ELISA assay used was previously described.⁴² (link) Briefly, recombinant SARS-CoV-2 RBD proteins or BSA (2.5 μg/mL) as negative control were prepared in PBS and adsorbed to plates overnight at 4°C. Coated wells were subsequently blocked with blocking buffer and then washed. CR3022 mAb (50 ng/ml) or a ½50 dilution of plasma from HD, or CI donors were prepared in a diluted solution of blocking buffer and incubated with the RBD-coated wells. Plates were washed followed by incubation with the respective secondary Abs. The binding of CR3022 IgG was quantified with HRP-conjugated antibodies specific for the Fc region of human IgG and used to normalize the RLU from each plate. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences). Light emission was measured with an LB942 Tri-Star luminometer (Berthold Technologies). Signal obtained with BSA was subtracted for each plasma and was then normalized to the signal obtained with CR3022 present in each plate. The seropositivity threshold was established using the following formula: mean of pre-pandemic SARS-CoV-2 negative plasma + (3 SD of the mean of pre-pandemic SARS-CoV-2 negative plasma).

Sannier G., Nicolas A., Dubé M., Marchitto L., Nayrac M., Tastet O., Chatterjee D., Tauzin A., Lima-Barbosa R., Laporte M., Cloutier R., Sreng Flores A.M., Boutin M., Gong S.Y., Benlarbi M., Ding S., Bourassa C., Gendron-Lepage G., Medjahed H., Goyette G., Brassard N., Delgado G.G., Niessl J., Gokool L., Morrisseau C., Arlotto P., Rios N., Tremblay C., Martel-Laferrière V., Prat A., Bélair J., Beaubien-Souligny W., Goupil R., Nadeau-Fredette A.C., Lamarche C., Finzi A., Suri R.S, & Kaufmann D.E. (2023). A third SARS-CoV-2 mRNA vaccine dose in people receiving hemodialysis overcomes B cell defects but elicits a skewed CD4+ T cell profile. Cell Reports Medicine, 4(3), 100955.

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Soluble gp120 Interaction with CD4

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The capacity of soluble gp120 produced in absence (DMSO) or presence of temsavir to interact with CD4 was evaluated by indirect ELISA. Soluble gp120 or bovine serum albumin (BSA) (0.1 μg/mL) as a negative control, were prepared in PBS and adsorbed to plates (MaxiSorp; Nunc) overnight at 4°C. Coated wells were subsequently blocked with blocking buffer (Tris-buffered saline [TBS] containing 0.1% Tween20 and 2% BSA) for 1h at room temperature. Wells were then washed four times with washing buffer (Tris-buffered saline [TBS] containing 0.1% Tween20). Primary Abs (CD4-Ig, A32 and C11) were diluted in blocking buffer and incubated with protein-coated wells for 90 min at room temperature. Plates were washed four times with washing buffer followed by incubation of HRP-conjugated secondary antibody specific for the Fc region of human IgG (Thermo Fisher Scientific) for 60 min at room temperature. The wells were then washed four times with washing buffer. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences). Light emission was measured with a LB942 TriStar luminometer (Berthold Technologies). The signal obtained with CD4-Ig was normalized with the signal obtained with A32 or C11.

Orihuela C.J., Mills J., Robb C.W., Wilson C.J., Watson D.A, & Niesel D.W. (2001). Streptococcus pneumoniae PstS Production Is Phosphate Responsive and Enhanced during Growth in the Murine Peritoneal Cavity. Infection and Immunity, 69(12), 7565-7571.

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Sandwich ELISA for Soluble gp120

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The concentration of soluble gp120 in the supernatant of gp160-expressing cells or after dialysis, filtration and concentration process was determined by anti-gp120 sandwich ELISA. The combination of nnAbs 17b and A32 were prepared in PBS and adsorbed to plates (MaxiSorp; Nunc) overnight at 4°C. Coated wells were subsequently blocked with blocking buffer (Tris-buffered saline [TBS] containing 0.1% Tween20 and 2% BSA) for 90 min at room temperature. Wells were then washed four times with washing buffer (Tris-buffered saline [TBS] containing 0.1% Tween20). Supernatant from gp 160-expressing cells were incubated with the nnAbs-coated wells for 120 min at room temperature. Plates were washed four times with washing buffer followed by incubation with HRP-conjugated C11 nnAbs (3 μg/mL) for 90 min at room temperature. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences, Waltham, MA, USA). Light emission was measured with a LB942 TriStar luminometer (Berthold Technologies, Bad Wildbad, Germany). A standard curve using known concentrations of purified recombinant gp120_YU2 was used to quantify the precise concentration of soluble gp120 in each supernatant.

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SARS-CoV-2 RBD ELISA Assay Protocol

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The SARS-CoV-2 RBD ELISA assay was used to measure the level of RBD-specific IgG, as previously described (Beaudoin-Bussieres et al., 2020 (link); Prevost et al., 2020 (link)). Briefly, recombinant SARS-CoV-2 RBD protein was prepared in PBS (2.5 μg/mL) and adsorbed to plates overnight at 4°C. Coated wells were subsequently blocked with blocking buffer then washed. CR3022 monoclonal Ab (50 ng/mL) at 1/250, 1/500, 1/1,250, 1/2,500, 1/5,000, 1/10,000, 1/20,000 dilutions of plasma from SARS-CoV-2-naive or previously infected donors were prepared in a diluted solution of blocking buffer and incubated with the RBD-coated wells. Plates were washed followed by incubation with the respective secondary Abs. Area Under the Curve (AUC) was calculated by using GraphPad. To calculate the RBD-avidity index, we performed a stringent ELISA where the plate was washed with washing buffer supplemented 8M urea. The binding of CR3022 IgG and plasma was quantified with HRP-conjugated antibodies specific for the Fc region of human IgG. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences). Light emission was measured with a LB942 TriStar luminometer (Berthold Technologies).

Nayrac M., Dubé M., Sannier G., Nicolas A., Marchitto L., Tastet O., Tauzin A., Brassard N., Lima-Barbosa R., Beaudoin-Bussières G., Vézina D., Gong S.Y., Benlarbi M., Gasser R., Laumaea A., Prévost J., Bourassa C., Gendron-Lepage G., Medjahed H., Goyette G., Ortega-Delgado G.G., Laporte M., Niessl J., Gokool L., Morrisseau C., Arlotto P., Richard J., Bélair J., Prat A., Tremblay C., Martel-Laferrière V., Finzi A, & Kaufmann D.E. (2022). Temporal associations of B and T cell immunity with robust vaccine responsiveness in a 16-week interval BNT162b2 regimen. Cell Reports, 39(13), 111013.

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