Sensitivity and applications of the nondenaturing staining of proteins on polyvinylidene difluoride membranes with Amido black 10B in water followed by destaining in water.

how sensitively proteins on PVDF can be detected with Sensitivity and Applications of the Amido black 10B dissolved in water and whether proNondenaturing Staining of Proteins on tein desorption from PVDF blots is impaired by stainPolyvinylidene Difluoride Membranes with ing and destaining in alcoholic and acidic solutions. Amido Black 10B in Water Followed by We tested, repeatedly, the sensitivities of various staining methods, using BSA dot blotted onto PVDF Destaining in Water strips (Immobilon P, Millipore) (Fig. 1). The membranes were dried, wet with methanol, rinsed in water, Fabrizio Gentile, Ernesto Bali, and stained, under shaking, for 5 min as follows: (i) and Giuseppe Pignalosa staining with 0.1% (w/v) Coomassie brilliant blue RCentro di Endocrinologia e Oncologia Sperimentale del 250 (Bio-Rad) in 50% (v/v) methanol, destaining with Consiglio Nazionale delle Ricerche and Dipartimento di two 3to 4-min changes of 50% methanol and 10% Biologia e Patologia Cellulare e Molecolare, Universita acetic acid; (ii) staining with 0.1% Amido black 10B ‘‘Federico II’’, Via S. Pansini 5, 80131 Naples, Italy (Carlo Erba) in 50% methanol, destaining with two 2to 3-min changes of 50% methanol and 10% acetic acid or of distilled water; (iii) staining with 0.1% Amido Received September 19, 1996 black 10B in distilled water, destaining with several changes of distilled water for 15 min; and (iv) staining The most widely used stains for proteins immobilized with 0.5% Ponceau S (Carlo Erba) in 1% acetic acid, on polyvinylidene difluoride (PVDF) membranes, e.g., destaining with several changes of distilled water for Coomassie brilliant blue R-250 (C.I. No. 42660) and 2 min. A Kodak amber filter was used to photograph Amido black 10B (C.I. No. 20470), are usually prepared membranes stained with Coomassie brilliant blue Rin water/alcohol (methanol or isopropanol) with or 250 and Amido black 10B, and a Kodak No. 11 Wratten without acetic acid (1–4). Destaining is also usually gelatin filter (green) was used for those stained with done in methanolic and acidic solutions (1, 3, 4). Water Ponceau S. Coomassie brilliant blue R-250 and Amido has been used to destain PVDF blots stained with black 10B, either in 50% methanol or in water, had Amido black 10B in water/alcohol/acid (2) or, in an atsimilar sensitivities, detecting concentrations as low as tempt to improve subsequent protein desorption, with 100–50 ng (8–4 ng/mm) of BSA. These values are in Amido black 10B in water (5). However, as yet, the good agreement with previous reports (1). Both disstaining sensitivities of aqueous and of methanolic and tilled water and 50% methanol, 10% acetic acid permitacidic solutions of Amido black 10B have not been comted rapid background destaining of blots stained with pared; similarly, no comparison has been made of the efficiencies of protein desorption from PVDF blots stained with the two methods. Protein elution from nitrocellulose blots is prevented by Coomassie blue staining in water/methanol/acetic acid (6), but not by staining with 0.5% Ponceau S (C.I. No. 27195) in 1% acetic acid, followed by destaining in water (7). The issue is far less clear-cut regarding protein recovery from PVDF. A 75% recovery was reported both for Ilabeled protein A eluted with acetonitrile from PVDF blots stained with 0.5% Ponceau S in 1% acetic acid (8) and for I-labeled bovine serum albumin and ovalbumin eluted with detergents from PVDF blots stained with Coomassie brilliant blue R-250 in water/methanol/acetic acid (9). However, 90–100% efficiencies, assessed by visual inspection and densitometry, were reported for the elution of BSA with detergents from FIG. 1. Sensitivity of four staining methods for BSA dot blotted PVDF blots stained with aqueous Amido black 10B and onto PVDF. The indicated amounts of BSA in 10 ml of water were dot blotted onto PVDF (Immobilon P, Millipore) and treated as foldestained in water (5). Consequently, we investigated lows: CB, stained with 0.1% (w/v) Coomassie brilliant blue R-250 in 50% (v/v) methanol and destained with 50% methanol and 10% (v/ v) acetic acid; ABM, stained with 0.1% (w/v) Amido black 10B in 50% 1 To whom correspondence should be addressed at Centro di Endocrinologia e Oncologia Sperimentale del C.N. R. , c/o Dipartimento di methanol and destained with 50% methanol and 10% acetic acid; ABH, stained with 0.1% Amido black 10B in distilled water and deBiologia e Patologia Cellulare e Molecolare, Universita ‘‘Federico II,’’ Via S. Pansini 5, 80131 Naples, Italy. Fax: (39-81) 7701016. stained with water; PR, stained with 0.5% (w/v) Ponceau S in 1% acetic acid and destained with water. The diameters of the dots 2 Abbreviations used: BSA, bovine serum albumin; PVDF, polyvinylidene difluoride. ranged between 3.5 and 4 mm.