WesternBright ECL requires up to ten times less antibody than other substratesĭuplicate slot blots containing serial dilutions of transferrin were probed with the antibody diutions shown, and detected with either Westernbright ECL or Pierce ECL (Thermo Scientific) according to the manufacturer’s instructions. Each blot was exposed to film for 1 minute. Duplicate Western blots with serial dilutions of HeLa cell lysate were probed for ERK1 and detected using either WesternBright ECL or Amersham ECL HRP substrates. The higher sensitivity of WesternBright ECL is especially important when detecting low abundance proteins. Westernbright ECL has high sensitivity for detection of low-abundance proteins WesternBright ECL is several times more sensitive than Amersham ECL. Both blots were simultaneously exposed to the same sheet of film for 15 seconds. Duplicate slot blots containing serial dilutions of transferrin protein were detected using WesternBright ECL or Amersham™ ECL™ (GE Healthcare). WesternBright ECL substrate produces a strong, long-lasting signal after reaction with HRP. WesternBright ECL is specially formulated to produce a strong signal for sensitive film imaging. WesternBright ECL is optimized for chemiluminescent Western blots imaged using X-ray film. Additionally, the WesternBright ECL signal is long lasting, allowing multiple exposures without substantial signal decay. WesternBright ECL is more sensitive than other chemiluminescent substrates and uses up to 10 times less antibody than other substrates, allowing you to save precious antibodies and samples. M, Magic Mark XP western protein standard, 2 µl/lane (Invitrogen, Carlsbad, CA).WesternBright ® ECL is a horseradish peroxidase substrate optimized for chemiluminescent Western blots imaged using X-ray film. All images were unaltered for brightness and contrast. (C and D) Both immunoblots in (A and B) were set-aside for 1 hr at which time they were re-exposed for 10 min. An immunoreactive band corresponding to actin (42 kDa) was detected in both instances. Both blots were exposed for 1 min in a FujiFilm LAS-4000 mini luminescent image analyzer (GE Life Sciences, Piscataway, NJ). (A and B) Immunoblots incubated in commercial ECL reagent (A) and self-prepared ECL reagent (B) as described above. The membrane was cut into two halves, and each half was incubated in ECL reagent. After washing, the membrane was incubated in bovine anti-goat IgG-HRP, followed by extensive washing as described above. The nitrocellulose membrane was incubated in anti-actin IgG for ∼3 hr at R.T. with gentle agitation on an orbital shaker, followed by a 10-min washing step in which the wash buffer was changed five times to remove all traces of non-fat milk. Thereafter, the nitrocellulose membrane was blocked in 5% non-fat milk (w/v) dissolved in wash buffer for 1 hr at R.T. Increasing concentrations of protein ranging from 1 to 50 µg total protein (protein estimation was performed by using the Pierce BCA protein assay kit Thermo Scientific, Rockford, IL) were resolved by SDS-PAGE under reducing conditions and transferred onto a nitrocellulose membrane (BIO-RAD, Hercules, CA) for 4 hr. We also include some helpful tips on how to avoid high background during immunoblotting.Ī comparison between commercial and self-prepared ECL reagents. 1) and present this protocol which is based on an earlier publication but with minor modifications, 6 hoping that it is useful for investigators in the field. In this technical report, we summarize our findings ( Fig. After scanning the literature carefully, testing chemicals from different vendors and trying different experimental conditions, we show that there exists an excellent alternative to commercially-available enhanced chemiluminescence kits. Consequently, these pricey kits can put enormous strain on any investigator managing a multi-person lab but with a limited supply budget. The most popular method used to visualize a protein at the nanogram level involves enhanced chemiluminescence, and several easy-to-use detection kits are commercially available to accomplish this goal, except that these kits are expensive and reagents have a relatively short shelf-life. Thus, it is a technique used by a vast number of laboratories, including ours, 2 – 5 to generate meaningful and interpretable results with relative ease. 1 For example, immunoblotting can be used to effectively assess changes in the protein level of any target gene following a specific cellular treatment, following overexpression or knockout/knockdown of a gene or following co-immunoprecipitation to study different scientific disciplines including spermatogenesis. Immunoblotting (i.e., western blotting) is a powerful technique used to detect a specific protein within a given sample or set of samples.
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