The reversible Zinc stain is a widely used staining method for the detection of proteins fractionated by polyacrylamide gels or SDS denatured gels. The Zinc reverse staining exploits the ability of biopolymers to bind Zn2+ and that of imidazole to react with unbound Zn2+ to produce insoluble zinc-imidazolate (ZnIm2). This zinc-imidazole complex deposits along the gel surface forming a deep white stained background, against which unstained biopolymer bands are visualized. The method is reversible; therefore, it reduces the risk of protein modification allowing the elution of reverse stained proteins used in biological and enzymatic assays. The proteins identified by Zinc staining could also be quickly processed for microsequencing or mass spectrometric analysis.
- Melting point: 68 K (419.53 °C, 787.15 °F)
- Boiling point: 1180 K (907 °C, 1665 °F)
- Density (near r.t.): 14 g/cm3
- Heat of fusion: 7.32 kJ/mol
- Heat of vaporization: 115 kJ/mol
- Molar heat capacity: 25.470 J/(mol·K)
The reversible Zinc staining technique is based on the interaction of Zinc ions (Zn2+) with biomolecules and proteins. The stain deposits the zinc metal precipitates in the gel, which turns the gel opaque white, while the sodium dodecyl sulfate coating on the proteins prevents the stains from binding to the proteins. This produces a negative image that enables the detection of clear protein bands against a semiopaque white polyacrylamide background.
Protocol (Fernandez-Patron C. et al. 1992)
- Briefly rinse the gel with double distilled water.
- Shake the gel in 0.2M imidazole for 5-10 minutes.
- Discard the imidazole solution.
- Shake the gel in 0.3M ZnCl2 for 30 seconds.
- Discard the zinc solutions and wash the gel with double distilled H2
- Visualize the gel against a dark surface.
Note: Protein bands appear transparent against the white background.
- Shake the gel in 2% citric acid for 5-10 minutes. Change the solution 2-3 times.
- The gel could be used for immunoblotting assays.
Reversible protein staining with zinc (Sasse. & Gallagher., 2003)
- Place the polyacrylamide gel in a plastic container containing 10 gel volumes of E-Zinc staining solution.
- Incubate with gentle agitation for 10 minutes.
- Replace the E-zinc solution with 10 gel volumes of E-zinc developer solution.
- Incubate for 2 minutes with gentle agitation.
- Replace the E-zinc developer solution with 10 gel volumes of distilled water.
- Incubate the gel with gentle shaking for 60 minutes.
- Change the water in the container.
- Visualize the protein bands on a dark surface.
- Incubate the gel with 10 gel volumes of E-Zinc eraser solution for 5-10 minutes.
- Wash the gel with distilled water.
Detection and purification of L1 capsid protein in E.coli (Javanzad. et al. 2013)
L1 major capsid protein is the significant molecule that assembles into virus-like particles of the human papillomavirus (HPV). In the study, the recombinant HPV L1 was expressed and purified using the E. coli system. The L1 protein was expressed in a fused form in an inducible expression system. The recombinant GST-L1 fusion protein was fractionated as an 82 kDa protein on SDS-PAGE. These L1 proteins were purified by Zn+2 reverse staining. The presence of an 82 kDa band for GST-L1 protein was then confirmed using the anti-HPV16 L1 monoclonal antibody Camvir 1. Zinc-reverse staining was found to be a sensitive and useful staining technique for the detection of viral proteins.
Obtaining protein sequence information (Castellanos-Serra. & Hardy., 2001)
Imidazole-zinc staining is a sensitive procedure for protein detection on the membrane after transfer as compared to the amido-black dye. The proteins fractionated on SDS-PAGE and identified using the zinc stain are then assayed for immunoblotting. This allows to obtain the sequence information from protein amounts that were detectable on the gel but insufficient for detection on the membrane after blotting.
Recovery of nucleic acids
The imidazole-zinc staining is also used for the detection and nondestructive micro purification of nucleic acids in recombinant DNA experiments. The nucleic acids are cloned, digested with restriction endonucleases, amplified with PCR, radioactively labeled, sequenced, and then analyzed by mass spectrometry. The detection limit of the method is similar to that of the UV irradiation of ethidium bromide-stained dsDNA. The applicability of this negative staining for nucleic acids has enabled the analysis of a variety of samples over a wide range of molecular masses (up to 10 kbp).
The zinc staining has been widely used for sample preparation for high-sensitivity mass spectrometry analyses of protein digests. It has been applied in proteomics for the detection on one-or two-dimensional gels. The reverse zinc staining has been found superior to silver staining because of the efficient recovery of tryptic peptides. Proteins can be successfully purified and quantified using the zinc-imidazole staining for molecular and proteomic analysis.
- Wear proper gloves and lab coats while handling the zinc stain.
- Extended incubation in zinc solution causes indiscriminate staining of the gel surface, leading to an inappropriate detection of the protein bands.
- Do not dry the gels stained with zinc-imidazole.
- The staining method relies on the presence of SDS in the gel; therefore, it is not suitable for native gels.
- Use 1.5 mm thick gels to obtain a bright white background for a good contrast.
Strengths and limitations
- The zinc staining technique is based on the selective precipitation of a white imidazole-zinc complex formation along with the gel. The stain is known for its high sensitivity, ease of use, and cost-effectiveness.
- Reverse zinc staining enhances the detection by detecting the undetected proteins in the gels already stained with Coomassie Blue.
- The method could also be used for the identification of glycolipids and viral proteins.
- The technique has a wide array of applications in the fields of proteomics, molecular biology, and spectrometry.
- The method is less sensitive than the Coomassie and colloidal Coomassie staining techniques.
- Fernandez-Patron C. et al. (1992), Biotechniques 12:564-573
- Sasse., & Gallagher., R. S. (2003). Staining proteins in gels. Curr Protoc Mol Biol, Chapter 10:Unit 10.6.
- Castellanos-Serra., & Hardy., E. (2001). Detection of biomolecules in electrophoresis gels with salts of imidazole and zinc II: a decade of research. Electrophoresis, 22(5), 864-73.
- Javanzad., A. Bolhassani., F. Doustdari., M. Hashemi., & Movafagh., A. (2013). Reverse staining method of polyacrylamide gels by imidazole-zinc salts for detection and purification of L1 capsid protein in E.coli. Journal of Paramedical Sciences (JPS), 4(2).