Tinciones de gel de proteínas SYPRO™

No. Proteins stained with SYPRO Ruby protein gel stain cannot be blotted onto membranes. The fixation step and the fixative-like solution that the dye is dispersed in prevents efficient blotting of proteins onto membranes.

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Basic protocol:
The basic protocol is optimized for standard 1 mm thick, 8 cm x 8 cm SDS-PAGE minigels, such as NuPAGE Invitrogen Bis-Tris and Tris-acetate gels, Invitrogen Tris-glycine gels, and Invitrogen Tricine gels.

1) Fix. After electrophoresis, place the gel into a clean container with 100 mL of fix solution (50% methanol, 7% acetic acid) and agitate on an orbital shaker for 30 min. Repeat once more with fresh fix solution.

2) Stain. Add 60 mL of SYPRO Ruby gel stain. Agitate on an orbital shaker overnight.

3) Wash. Transfer the gel to a clean container and wash in 100 mL of wash solution (10% methanol, 7% acetic acid) for 30 min. The transfer step helps minimize background staining irregularities and stain speckles on the gel. Before imaging, rinse the gel in ultrapure water a minimum of 2 times for 5 min to prevent possible corrosive damage to the imager.


Rapid protocol:
The rapid protocol is optimized for standard 1 mm thick, 8 cm x 8 cm SDS-PAGE minigels, such as NuPAGE Invitrogen Bis-Tris and Tris-acetate gels, Invitrogen Tris-glycine gels, and Invitrogen Tricine gels.

1) Fix. After electrophoresis, place gel into a microwavable container with 100 mL of fix solution and agitate on an orbital shaker for 15 min Repeat once more with fresh fix solution.

2) Stain. Add 60 mL of SYPRO Ruby gel stain. Microwave 30 seconds, agitate 30 seconds to distribute heat evenly, microwave another 30 seconds to 80-85°C, and agitate on an orbital shaker for 5 min. Reheat the gel by microwaving a third time for 30 seconds and then agitate on an orbital shaker for 23 min for a total stain time of 30 min. An acceptable alternative to the microwave procedure is to incubate the gel at 80°C in a shaking water bath for 30 min.

3) Wash. Transfer the gel to a clean container and wash in 100 mL of wash solution for 30 min. The transfer step is necessary to avoid heating the destain solution, which may reduce stain sensitivity and also helps minimize background staining irregularities and stain speckles on the gel. Before imaging, rinse the gel in ultrapure water a minimum of 2 times for 5 min to prevent possible corrosive damage to the imager.

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Your samples or the gel wells were contaminated with keratins from skin or hair. Rinse out the gel wells with ultrapure water or running buffer before loading samples. Wear a lab coat and gloves when preparing samples and use microfuge tubes that have been stored in sealed plastic bags, not left out on the bench top, for preparing samples.

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Blue-colored dyes absorb light in the red wavelengths, so they absorb the red fluorescent emission of SYPRO Ruby dye. SYPRO Ruby dye still binds these proteins, but the signal is quenched by the colored dye, resulting in a negatively stained, dark band. Examples of molecular weight markers with blue-colored proteins that will quench SYPRO Ruby fluorescence are the BenchMark Pre-Stained Protein Ladder and some proteins in the SeeBlue Plus2 Pre-Stained Standard. The same phenomenon can be seen with the bromophenol blue dye front, if it is not completely run off the gel, and loss of signal when SYPRO Ruby stained gels are subsequently stained with Coomassie Blue stains. Most other colored dyes do not quench the SYPRO Ruby dye signal and will appear as normally stained protein bands.

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Speckles on the gel can increase as the SYPRO Ruby Protein Gel Stain ages, due to self-aggregation of the SYPRO Ruby dye over time. Speckles can also form due to dye aggregation around contaminants from the staining container, solutions, or particles from the air or gloves, including keratin proteins from skin and hair. When gels are incubated with SYPRO Ruby Protein Gel Stain for several hours or longer, dye can build up on the sides of the staining container and then be dislodged with continuing rocking, especially during the destain step, forming speckles. Non-dye speckles can also show up in the image from auto-fluorescent particles of dust, hair, glove powder, or clothing lint that falls on the gel or surface of the glass imaging plate. The better the imager is at focusing on surface features of the gel, the more speckles that are going to be visible.

To minimize the formation of speckles and other background debris, follow clean laboratory practices, use ultrapure water of greater than 18 megohm-cm resistance to prepare solutions, rinse gloves in water to remove powder residue before touching gels, use lint-free wipes and wear a lab coat or avoid wearing clothing that generates a lot of lint, always rinse the staining container with ethanol and wipe out any residual dye before staining another gel, and always rinse and wipe down the glass imaging surface with ethanol and water before placing your gel down. Remove dye buildup on the surface of the staining dish by wiping out the dish with ethanol between the stain and wash step. The rapid stain protocol is complete in as little as 90 minutes, which does not allow enough time for most speckles for form. Once speckles have been deposited on the gel, it is not possible to wash them off. Speckles will show up as sharp, tall spikes on 3D renditions of gel images. These spikes look distinct from 3D renditions of protein spots or bands. Some image analysis software packages have de-speckling algorithms that can easily identify and remove this type of pixelated noise.

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