SYPRO™ Protein Gel Stains

No. Dye concentrations higher than 1X do not give better detection. Instead, background fluorescence increases and, as the dye concentration increases, the dye becomes self-quenching and the signal actually decreases.

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Gels stained with SYPRO dyes can be dried between sheets of cellophane, although there is sometimes a slight decrease in sensitivity. If the gels are dried onto paper, the light will scatter and the sensitivity will decrease. Other plastics are not recommended, as the plastic typically used is not transparent to UV light.

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Yes, you may use SYPRO Orange Protein Gel Stain with Invitrogen gels but the protocol would have to be modified. The stain is mainly to be used with 0.05% SDS running buffer while all our buffers contain 0.1% SDS. This results in high background as the SDS binds tightly to the stain. The following protocol is recommended for best results with Invitrogen gels:

Wash solution: 7.5% (v/v) Acetic acid
Staining solution: 1:5000 SYPRO Orange in Wash solution

After the gel run is over, wash the gel in 100 ml of Wash solution for 10 minutes.
Place gel in Staining solution for up to 24 h in the dark.
Remove the gel from the Staining solution and rinse briefly in 100 ml Wash solution.
The gel is then ready to be photographed.

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No. Loading solutions contain so much SDS that SYPRO Ruby, SYPRO Orange and SYPRO Red dyes simply localize in the free SDS and bind very little of the proteins. Proteins can be covalently pre-labeled with ATTO-TAG CBQCA (Cat. No. A2333), DDAO succinimidyl ester (Cat. No. C34553) or TAMRA-succinimidyl ester (Cat. No. C2211) dyes, or the TC-FLAsH Expression Analysis Detection Kits (Cat. No. A10067 for orange fluorescence, Cat. No. A10068 for red fluorescence) prior to electrophoresis without affecting protein migration through the gel.

SYPRO Orange or SYPRO Red Protein Gel Stain can be diluted 5000-fold into the cathode (upper) buffer tank to stain proteins during electrophoresis without affecting migration. The problem with doing this is that there is considerable background fluorescence in the gels from the dye interacting with SDS. This can be reduced after electrophoresis by destaining in 7.5% acetic acid for 15-60 minutes. This method also results in poorer protein sensitivity than the standard post-staining method, requires the same amount of time before the gel can be imaged, and contaminates the electrophoresis apparatus.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.