Samples were prepared in RIPA buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM EDTA (pH 8.0), 1% NP-40 (Nacalai Tesque), 1% SDS (Nacalai Tesque), 1% sodium deoxycholate (Sigma–Aldrich), supplemented with cOmplete, Mini (Roche)) or RIP Lysis buffer (20 mM Tris-HCl (pH 7.4), 100 mM KCl, 1.5 mM MgCl2, 0.5% NP-40, supplemented with cOmplete, Mini (Roche)). Samples were mixed with loading buffer containing β-mercaptoethanol, boiled at 95 °C for 5 min, and cooled on ice. Samples were loaded on 5–20% polyacrylamide gels (ATTO), electrophoresed, and transferred to PVDF membranes (Bio-Rad). For BRCA2 and FANCM proteins, samples were loaded on NuPAGE 3–8% Tris-Acetate Protein Gels (Thermo Fisher Scientific). The membranes were immersed in 5% skim milk (BD Difco) in TBS-T. Signal Enhancer HIKARI for Western Blotting and ELISA (Nacalai Tesque) or 5% skim milk in TBS-T was used to dilute primary and secondary antibodies. Blots were developed using Luminata Forte Western HRP Substrate (Millipore) according to the manufacturer’s instructions. Chemiluminescent detection was performed using Amersham Imager 600 (GE Healthcare).
Signal enhancer hikari for western blotting and elisa
Manufactured by Nacalai Tesque
The Signal Enhancer HIKARI is a laboratory product designed to improve the detection sensitivity of Western Blotting and ELISA assays. It is a solution that can be applied to enhance the signals generated during these analytical procedures, without making any claims about its intended use or performance.
Automatically generated - may contain errors
Lab products found in correlation
Skim milk, by Nacalai Tesque (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
Np 40, by Roche (1 mentions)
Sodium dodecyl sulfate (sds), by Nacalai Tesque (1 mentions)
Np 40, by Nacalai Tesque (1 mentions)
Nupage 3 8 tris acetate protein gel, by Thermo Fisher Scientific (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Chemidoc touch mp, by Bio-Rad (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
Chemi lumi one ultra, by Nacalai Tesque (1 mentions)
2 protocols using signal enhancer hikari for western blotting and elisa
1
Western Blot Analysis of BRCA2 and FANCM
2
Quantifying Transgene Expression in C. elegans
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To check the expression level of transgenes, 15 one-day adult worms were collected in 10 µL of distilled water and flash-frozen in liquid nitrogen. The sample was then thawed and mixed with 3 µL of 4× SDS-PAGE sample buffer and 1.2 µL of 10% NP-40 and was boiled at 98°C for 5 min.
The samples were loaded onto a 5-20% gradient SDS-PAGE gel (Wako, Osaka, Japan)
and transferred to an Immobilon-P Transfer Membrane (Merck). Antibodies were diluted with Signal Enhancer HIKARI for Western Blotting and ELISA (Nacalai Tesque). Primary antibodies rabbit anti- SPD-5 (1:2000) and rat anti-GFP (1:10,000) were generated against full-length recombinant proteins (MBL Life Science). Additional primary antibodies were mouse anti-GST RRID AB_2340639). Signals were detected using Chemi-Lumi One Ultra (Nacalai Tesque), and blot images were obtained with a ChemiDoc Touch MP (Bio-Rad, California, USA).
The samples were loaded onto a 5-20% gradient SDS-PAGE gel (Wako, Osaka, Japan)
and transferred to an Immobilon-P Transfer Membrane (Merck). Antibodies were diluted with Signal Enhancer HIKARI for Western Blotting and ELISA (Nacalai Tesque). Primary antibodies rabbit anti- SPD-5 (1:2000) and rat anti-GFP (1:10,000) were generated against full-length recombinant proteins (MBL Life Science). Additional primary antibodies were mouse anti-GST RRID AB_2340639). Signals were detected using Chemi-Lumi One Ultra (Nacalai Tesque), and blot images were obtained with a ChemiDoc Touch MP (Bio-Rad, California, USA).
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