Coomassie staining is a simple and relatively sensitive method for visualizing separated proteins in polyacrylamide gels. The gel is soaked in a Coomassie R250-containing dye solution containing 40% methanol and 7% acetic acid. Excess stain is then removed by destaining the gel in 40% methanol/7%acetic acid until the background is clear. Since Coomassie blue binds non-specifically and stoichiometrically to all proteins, it can be used to determine relative amounts of proteins by densitometry for quantification.
The Colloidal Blue Staining Kit from Invitrogen offers a Coomassie blue G250-based colloidal staining with higher sensitivity than traditional Coomassie staining: it can detect down to 10 ng protein. The method is based on staining with Colloidal blue dye in methanolic solution with phosphoric acid and high ammonium sulfate concentration. The hydrophobicity of the Colloidal blue solution reduces the amount of free dye in the solution, which results in a higher affinity of the dye for proteins and lower background staining. The kit is recommended for staining of NuPAGE Novex-Tris Acetate, Tris-Glycine, Tricine and Zymogram pre-cast gels from Invitrogen, but also works well with homemade SDS Tris-Glycine gels. In comparison to the methanol/acetic-based destain solution during Coomassie staining, the Colloidal blue staining only requires water for destaining. The kit contains two solutions, 250 ml of Stainer A and 50 ml of the dye-containing Stainer B; both are stable for one year at room temperature. The staining solution is prepared by mixing Stainer A and B with methanol and deionized water directly before use. When staining NuPage„¥ Novex Bis-Tris gels, a different staining protocol is recommended by the manufacturer. The volumes are dependent on the number of gels to be stained.
Different gels require different fixation and staining protocols (as described in the manual of the manufacturer). I used the kit most often for staining gels after protein transfer to Western blots in order to check transfer efficiency. To stain separated proteins in homemade SDS Tris-Glycine gels prior to mass spectrometry, I used ultrapure water and methanol. After separation of the proteins on 5 and 10% gels, I washed the gels briefly in deionized water, fixed them on a shaking platform for 10 to 30 min in a solution of 50% methanol/10% acetic acid (protocol for NuPage„¥ Novex Bis-Tris gels in the manual) and incubated them for 10 min in the solution containing 20% methanol, 20% Stainer A. Before adding, it is important to resuspend the settled dye in the Stainer B bottle to avoid staining with a less concentrated dye solution which will result in weaker staining. I added 5% of Stainer B and incubated the gels on a shaker for 4 hours or overnight before destaining them in deionized water (3-7 hours). For thicker gels (1.5 cm), a longer staining time (overnight) resulted in more intense staining, but longer (7 h to overnight) destaining procedures were necessary to reduce the background completely. Longer staining procedures also enhanced the visibility of larger protein bands. To avoid washing out proteins, especially lower molecular weight (10-30 kD) proteins, during longer staining/destaining procedures, I fixed the gels before staining. The background was higher in lower percentage (5%) gels as compared to higher percentage gels (10%, 12% gels) due to increased trapping of the Colloidal dye in the bigger pores. Longer destaining procedures are then necessary to completely destain the gel for quantification of the proteins.
In summary, the Colloidal Blue Staining Kit from Invitrogen is an easy to use, versatile and sensitive staining procedure for a variety of applications. For standard procedures, staining and destaining of the gels is fast (within 3-4 and 7 hours, respectively), but the times can be extended as necessary for better results in different gels. The manual provides sufficient information for the staining procedures of Invitrogen precast gels, but optimizing the staining for gels from different vendors, homemade gels or different applications needs to be performed by the users themselves.