Electrophoresis Protein Staining Reagents Selection Guide

Protein Dyes and Stains Selection Chart

The diversity of protein analysis methods require a wide selection of protein visualization (staining) reagents. EZBlue™ and ProteoSilver™ reagents are designed specifically for proteomics, but they also perform impressively in traditional PAGE formats.

Optimize your protein detection by choosing the best reagent for your application using the chart below.

Protein Stain Applications

Alcian Blue 8GX is a cationic dye that forms insoluble complexes with acidic glycosaminoglycans, thereby helping in the quantitative determination of glycosaminoglycans. It is used for staining bone and cartilage, sulfated proteoglycans in murine MPC (mesenchymal progenitor cell) nodule, and sulfated proteoglycans in bovine articular chondrocytes.

Amido black (AB) is an acidic dye that can detect proteins levels of 50 ng in both polyvinylidene difluoride (PVDF) and nitrocellulose membranes. AB aids protein visualization even at low concentrations and has applications in forensics due to its ability to stain blood proteins in fingerprints.

Brilliant Blue G is a methanol-based stain designed for polyacrylamide gels but will also stain protein in agarose gels (IEF). The stain contains methanol and acetic acid so gels do not require fixing before staining. This stain is also used for protein staining of cells on microscopic slides.

Brilliant Blue R staining solution is especially designed for use in staining protein in agarose gels and Ouchterlony gels. The stain contains ethanol and acetic acid so gels do not require fixing before staining.

Eosin Y disodium salt has been used as a counterstain in immunocytochemistry and immunohistochemistry. Eosin Y is a xanthene dye and is used for the differential staining of connective tissue and cytoplasm. In histopathology, it is applied as a counterstain after hematoxylin and before methylene blue. It is also used as a background stain, thereby giving contrast to the nuclear stains.

EZBlue™ Gel Staining Reagent is convenient and sensitive colloidal protein stain that improves protein electrophoresis results while significantly reducing staining time.. As a colloidal stain, it reacts only with proteins and not the gel. Protein bands can be visualized almost immediately. No destaining step is required, although a water wash may intensify bands and clarify the background. EZBlue^® Stain is extremely sensitive, detecting down to 5 ng of protein.

Fast Green FCF is a biocompatible triarylmethane dye. Fast Green FCF dye is used for protein staining in IEF, native PAGE and SDS-PAGE. Fast Green staining is linear over a wider range of protein concentrations. After electrophoresis, fixing the proteins in the gel is recommended for maximum sensitivity. The gel can be destained with either 30% (v/v) ethanol, 10% (v/v) acetic acid, or 7% acetic acid. The stained gel can be scanned at 625 nm. The detection sensitivity is approx. 30% that with Brilliant Blue R. Fast Green FCF has been used for in staining of menisci from rabbit and of osteochondral tissues.

Fixing Solution is an effective for pre-stain protein fixing, suitable for SDS-PAGE, non-denaturing PAGE and IEF. Proteins require fixing into the gel for analytical purposes so that they do not leach from the gel following electrophoresis.

Glycoprotein Detection Kit is for the selective staining of glycoproteins on polyacrylamide gels and membranes using a modification of the Periodic Acid-Schiff (PAS) method. Staining of sugar moieties of glycoproteins yields magenta bands with a colorless background. The periodic acid/Schiff reagent stains vicinal diol groups found mainly on peripheral sugars and sialic acids.

Colloidal Gold is used as a protein and polysaccharide marker for electron microscopy. It is also used as a general stain for protein immobilized on nitrocellulose membranes in spot tests or on blots prepared from polyacrylamide gels after electrophoresis.

Oil Red O stain is suitable as a lipid/lipoprotein stain on cellulose acetate. The dye begins precipitating during staining. After staining (1 hr), the membrane is destained in methanol:glycerol (1:3). Often cited for use with agarose.

Ponceau S is the sodium salt of a diazo dye. Ponceau S stain is supplied ready-to-use for rapid (5 min) staining of protein bands on nitrocellulose or PVDF membranes (Western blots) and also for staining protein on cellulose acetate membranes. Ponceau S staining on nitrocellulose and PVDF is reversible by washing the stained membrane with 0.1M NaOH for 1 minute.

ProteoSilver™ Silver Stain Kit is a stable, highly sensitive staining kit. The kit contains prepared solutions of silver staining reagents and detailed instructions to achieve optimal results. With a detection limit of 0.1 ng/mm² of protein (BSA) and an extremely low background, this stain can detect low-abundance proteins.

ProteoSilver™ Plus Silver Stain Kit is similar to ProteoSilver™ Silver Stain Kit (above). The ProteoSilver™ Plus is also MALDI-MS compatible and does not contain glutaraldehyde, which crosslinks lysine residues, resulting in inaccurate spectra. ProteoSilver™ Plus Silver Stain Kit contains two additional reagents for destaining an excised protein spot, for those wishing to perform further characterization through tryptic digestion and MALDI-MS analysis.

Reversible Protein Detection Kit for Membranes and Polyacrylamide Gels is a unique protein detection system designed for staining of proteins on nylon, nitrocellulose, and PVDF membranes or PAGE gels with the detection sensitivity similar to that of Coomassie stains. The staining is reversible with an EDTA solution so that the blot can be reused for Western blotting or for amino acid sequence analysis (PVDF). This kit does not contain the toxic chemicals cacodylic acid or potassium ferrocyanide.

Sudan Black B stains phospholipids and intracellular lipids. Staining pattern is similar to myeloperoxidase reaction, with positive staining of granulocytic cells, eosinophils and weak monocytic staining. Sudan Black B stains phospholipids gray and neutral fats blue-black.

SYPRO^® Orange is similar to SYPRO^® Red although it is somewhat brighter and gives slightly higher background fluorescence. For those using a laser-excited gel scanner, an argon-ion laser-based instrument is recommended for SYPRO^® Orange. The dye is efficiently excited by UV or broad band illumination. It is not suitable for staining proteins on blots or in IEF gels and shows reduced sensitivity when staining proteins on 2-D gels.

SYPRO^® Ruby protein gel stain is a ready-to-use, ultrasensitive, luminescent stain for proteins separated by PAGE. This stain, designed for use in 2-D PAGE, is also effective for 1-D PAGE and isoelectric focusing (IEF) gels. SYPRO^® Ruby protein gel stain attains sensitivity comparable to many silver stain techniques.