Cells were washed in PBS and lysed in 1× NuPAGE LDS Sample Buffer (Life Technologies). NuPAGE Reducing Agent was added to the samples prior to heating at 95°C. Proteins were separated on a NuPAGE Novex Bis-Tris 4-12% gel in a Bio-Rad system with MOPS SDS Running Buffer (Life Technologies). To transfer proteins from the gel onto an Immun-Blot PVDF Membrane (Bio-Rad), a wet blotting system (Bio-Rad) was used with NuPAGE transfer buffer containing 10% methanol. The membrane was blocked with 5% semiskimmed milk (Sigma), 0.1% Tween 20 (Calbiochem) in PBS (GIBCO). This was followed by incubation with the primary antibody against NFκB p65 (abcam, ab7970) in blocking buffer. After washing the membrane, the antibody swine-anti-rabbit with horseradish peroxidase (HRP) conjugate (Dako) was added. Subsequent to washing the membrane, HRP was visualized by ECL Lumilight Plus (Roche), and the signal was captured with the ImageQuant LAS4000 (GE Healthcare). Quantification of the protein bands in the images was done with ImageJ Fiji.
Ecl lumilight plus
Manufactured by Roche
The ECL Lumilight Plus is a chemiluminescent substrate solution used for the detection of proteins in Western blot analysis. It produces a luminescent signal when in contact with the horseradish peroxidase (HRP) enzyme, which can be captured and quantified using a compatible imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Ab7970, by Abcam (1 mentions)
Mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Immun blot pvdf membrane, by Bio-Rad (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Gradient sds page gel, by Bio-Rad (1 mentions)
2 protocols using ecl lumilight plus
1
Western Blot Analysis of NFκB p65
2
Western Blot Protein Detection Protocol
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Whole cell extracts were prepared in Laemmli buffer (Tris HCl 62.5mM, SDS1%, Mercaptoethanol 5%, glycerol 25% and bromophenol blue). Samples were sonicated and heated at 95°C for 5 minutes before their separation on a 4–15% gradient SDS PAGE gel (Biorad, 4–15%). Proteins were transferred from gel to nitrocellulose membrane using semi-dry method. Once blocked in PBS-Tween (5%) plus 10% skimmed milk, membrane were firstly immunoblotted with primary antibody diluted in 2% milk PBS-T (generally 1/1000 except for anti H3 whose dilution used is 1/10000) at 4°C overnight and finally immunobloted with secondary antibody coupled to HRP (Sigma) diluted in 2% milk PBS-T at room-temperature for 1 hour. After several washes in PBS-T, proteins were detected using ECL lumi-light plus (Roche) and images acquired using camera system (Chemitouch Biorad).
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