Comparative advantages of imidazole–sodium dodecyl sulfate–zinc reverse staining in polyacrylamide gels

Abstract

Polyacrylamide gel electrophoresis (PAGE), which is widely used to assess protein purity and molecular weight [1], is best combined with a highly sensitive, reproducible, inexpensive, rapid, and easy to perform protein staining/detection method. Coomassie brilliant blue (CBB) 1 staining is a popular method, known for its simplicity, economy, and compatibility with downstream analysis but relatively poor sensitivity (about 50 ng protein/band), low aYnity for acidic proteins, variability in background staining, and lack of reproducibility. The silver staining procedure is sensitive but relatively tedious and is not speciWc for proteins [2,3]. Rutheniumcomplex-based SYPRO Ruby Xuorescent staining is protein speciWc and as sensitive as silver staining. It has a broad linear dynamic range and a simple stain/destain protocol and is compatible with subsequent mass spectrometry or Edman sequencing [4,5]. Imidazole–SDS– zinc reverse staining is reportedly more sensitive than CBB staining [6] and approximately as sensitive as silver staining [2]. In this method a white, insoluble imidazole– zinc complex forms on the surface of gels as a background against which proteins, complexed with SDS during pretreatment, appear as unstained, transparent bands [7]. In the study reported here, a comparison of the imidazole–SDS–zinc, SYPRO Ruby, silver, and CBB staining methods was made. Imidazole–SDS–zinc reverse staining, with minor modiWcations of the original method [6,8], was shown to best meet all the optimal criteria (as stated above), while allowing sensitive, reproducible detection and sample recovery. To compare the sensitivity, reproducibility, and simplicity of staining methods, four Tris–glycine SDS–PAGE gels [9] were equally loaded, run under identical conditions, rinsed with distilled water, stained, and photographed using a VersaDoc 4000 Imager (Bio-Rad, California, USA). The gels were loaded with 500–1 ng/protein, from serial twofold dilutions of 1 mg/ml of phosphorylase b, bovine serum albumin (BSA), ovalbumin, and carbonic anhydrase. Additionally, three homogenates were applied, a puriWed neutrophil homogenate of »170,000 cells [10], a monocyte homogenate of »50,000 cells, and whole blood homogenate (»0.25l blood) [11]. CBB staining was carried out with some modiWcations of the method of Fernandez-Patron et al. [6]. Overnight staining [0.125% (m/v) CBB R–250, 50% (v/v) methanol, 10% (v/v) acetic acid] was followed by destaining for several hours in multiple changes of destain [50% (v/v) methanol, 10% (v/v) acetic acid], followed by rehydration [5% (v/v) methanol, 7% (v/v) acetic acid]. Gels were illuminated from below with white light and photographed without a Wlter. SYPRO Ruby Xuorescent staining was conducted according to the manufacturer’s instructions (Molecular Probes, Oregon, USA). Gels were Wxed, stained overnight, rinsed, placed on a UV-transilluminator (312 nm), and photographed using a 610-nm longpass emission Wlter. Imidazole–SDS–zinc reverse staining was eVected by pretreating gels in imidazole–SDS solution [200 mM imidazole, 0.1% (m/v) SDS, 10–15 min] and rinsing brieXy (distilled water, 30 s) before developing (200 mM zinc sulfate) until the gel background turned intensely white with transparent protein bands (15–60 s). The extent of development was best monitored during manual agitation of