Enhanced chemiluminescence (ECL) for routine immunoblotting

Abstract

Immunoblotting is an analytical technique used by many laboratories to study protein expression. It involves electrophoretic separation of proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immobilization of these proteins onto a membrane (i.e., nitrocellulose or polyvinylidene difluoride), incubation in a monoclonal or polyclonal antibody and detection by a standard method such as enhanced chemiluminescence (ECL). To achieve this, most laboratories opt to use commercially-available chemiluminescence kits which are acceptable but relatively expensive. In this technical report, we show that a self-prepared chemiluminescence reagent is superior to a commercially obtained kit in terms of sensitivity, duration of signal, ease-of-use and shelf-life but at a fraction of the cost of a kit.

Figures and Tables

Figure 1 A comparison between commercial and self-prepared ECL reagents. Increasing concentrations of protein [adult testis lysate prepared in lysis buffer: 50 mM Tris pH 7.4 at 22 °C containing 0.15 M NaCl, 2 mM EDTA, 1% NP-40 (v/v), 10% glycerol (v/v), protease and phosphatases inhibitor cocktails] 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. Thereafter, the nitrocellulose membrane was blocked in 5% non-fat milk (w/v) dissolved in wash buffer [PBS-Tris/0.1% Tween-20 (v/v)] for 1 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. The nitrocellulose membrane was incubated in anti-actin IgG [diluted 1:200 in PBS-Tris pH 7.4 at 22 °C containing 0.1% Tween-20 (v/v), 0.1% BSA (w/v) and 0.05% NaN3 (w/v); cat. no. sc-1616; lot no. K0510, Santa Cruz Biotechnology, Santa Cruz, CA] for ∼3 hr at R.T. with agitation. After washing, the membrane was incubated in bovine anti-goat IgG-HRP [diluted 1:2000 in PBS-Tris pH 7.4 at 22 °C containing 0.1% Tween-20 (v/v) and 0.1% BSA (w/v); cat. no. sc-2378; lot. no. D0910], followed by extensive washing as described above. The membrane was cut into two halves, and each half was incubated in ECL reagent. (A and B) Immunoblots incubated in commercial ECL reagent (A) and self-prepared ECL reagent (B) as described above. Both blots were exposed for 1 min in a FujiFilm LAS-4000 mini luminescent image analyzer (GE Life Sciences, Piscataway, NJ). An immunoreactive band corresponding to actin (42 kDa) was detected in both instances. (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. All images were unaltered for brightness and contrast. M, Magic Mark XP western protein standard, 2 µl/lane (Invitrogen, Carlsbad, CA).