| | Re: Protein Staining
To enable visualization of the migration of proteins it is common to include in the loading buffer a small anionic dye molecule. Anionic dyes have a negative charge and attach to cationic surfaces and vice versa when the cationc dyes which are positively charged attach to anionic surfaces. The anionic dyes are bromophenol blue, chromotrope 2R, Coomassie brilliant blue, naphthol yellow S, orange G, and sulforhodamine B (SRB). Since the dye is anionic and small, it will migrate the fastest of any component in the mixture to be separated and provide a migration front to monitor the separation progress.
Gel loading buffer type A : dissolve in 10 ml dH2O, 4 g sucrose and 25 mg bromophenol blue or xylene cyanol, mix filter or/and add preservative to avoid mould growing in the sucrose, and store at 4 °C, stable per 2 years.
Gel loading buffer type B : dissolve in 10 ml dH2O, 1.5 g di Ficoll 400 and 5 mg Orange G, mix and in small aliquots store at 4 °C or RT, stable per 2 years. To use, add and mix 1/5th volume of Gel loading buffer type B to DNA solutions prior to loading into the wells of gels.
Gel loading buffer type C : Sol. A : dissolve in 400 ml dH2O, 195 mM Tris adjuste at pH 6.8 at 25 °C with 1 N HCl, then dissolve 60 g SDS, add 400 ml dH2O and 300 ml glycerol, and dissolve 0.3 g bromophenol blue and fill 1 liter with dH2O. Mix and in small aliquots by 8 ml, and store at 4 °C, stable for 1 year; Sol. B : dissolve 962.5 mg (1.25 M) di Dithiothreitol (DTT m.w. 154) in 10 ml aqueous solution containing 59 mM mannitol (m.w. 182.17)and 18 mM EDTA disodium (m.w 372.20). The liquid is a buffered solution capable of maintaining the liquid at a physiological pH about 6.7 at about 25 °C. Mix and in small aliquots by 1 ml, and store at 4 °C, stable for 1 year. DTT is present to help reduce any disulphide S-S bonds that help form secondary/tertiary structure and/or dimer formation. The SDS detergent binds to the proteins positive charges so that proteins will separate based on size and not by charge. The SDS also denatures the proteins and subunits to also help separate them based on size, not on shape. To use, add 1/10 volume of the Sol. B to 1 volume Sol A. The add 1/2 volume of the above mix to the protein sample, and heat at 100 °C for 5 minutes to denature the protein. During protein sample treatment the sample should be mixed by vortexing before and after the heating step for best resolution. Spin for 30 sec. in a centrifuge at 10900 rpm to remove precipitated material, and load onto gel.