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Please check our Protein Gel and Membrane Stains website page: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-gel-electrophoresis/protein-gel-staining-imaging.html
Most of the stains we offer are compatible with Tris-Glycine gels. Dyes will react mainly with protein regardless of gel chemistry.

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Similarly to ethidium bromide, SYBR Safe DNA Gel Stain runs in the opposite direction of the migrating DNA. This has no practical effect on the use of gels cast with SYBR Safe DNA Gel Stain, as only the very bottom of the gel will have a lower concentration of stain. This effect can be partially counteracted by staining the gel with SYBR Safe DNA Gel Stain after electrophoresis. Solutions of dye should not be added to the running buffer as this can cause breakdown of the dye at the electrodes and release toxic volatile compounds into the air.

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Many factors could affect the intensity of the bands as summarized below.

(1) Insufficient RNA was loaded on the gel. Increase the amount of RNA loaded.

(2) RNA was degraded. Avoid nuclease contamination of the RNA standards. Store RNA at -70 degrees C in formamide. Deionize formamide and glyoxal, store aliquots at -20 degrees C.

(3) RNA was electrophoresed off the gel. Electrophorese the gel for less time, at a lower voltage, or in a higher percentage gel.

(4) For ethidium bromide-stained RNA, improper UV light source was used. Use short-wavelength (254 nm) UV light. For ethidium bromide-stained RNA, improper staining and destaining conditions were used. Stain in the dark with 5 mg/mL ethidium bromide. For thin (3.2 mm) formaldehyde gels, stain 5 min and destain 1 h. For thicker gels, stain 30 min and destain 2 hr. To reduce background staining, use 0.66 M formaldehyde instead of 2.2 M formaldehyde in gels. For glyoxal RNA gels, stain and destain in 0.5 M ammonium acetate to help reduce background staining.

(5) For radiolabeled RNA, an improper labeling method was used.

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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.

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

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Similar to ethidium bromide, SYBR Safe stain runs in the direction opposite migrating DNA. This has no practical effect on the use of precast gels containing SYBR Safe stain, as only the very bottom edge of the gel will have a lower concentration of stain. This effect can be counteracted in a standard gel run by restaining the gel after electrophoresis.

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Please go here (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/nucleic-acid-gel-electrophoresis/dna-stains/sybr-safe.html) and click on the “Filter Recommendations” tab to see filter recommendations for use with SYBR Safe DNA Gel Stain. Note that the excitation and emission spectra of SYBR Safe DNA Gel Stain are very similar to those of SYBR Green I, SYBR Green II, and SYBR Gold dyes, as well as fluorescein (FITC). Therefore, filters appropriate for these dyes can also be used. A camera filter is not required with the Safe Imager Blue-Light Transilluminator; the amber filter provided with the instrument serves this purpose.

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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.

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Detection sensitivity is only slightly diminished by dilution of SYPRO Ruby Gel Stain in water up to 5-fold. However, both the fluorescence signal as well as the dynamic range are both reduced significantly with even a 1/2 dilution. Detection sensitivity also remains high if the stain is reused up to two times, but signal intensity is reduced up to 2.5-fold in twice-used stain. Increasing the staining volume to 100 mL is recommended when reusing the stain.
Reference: Ahnert N, Patton WF, Schulenberg B. Optimized conditions for diluting and reusing a fluorescent protein gel stain. Electrophoresis 2004, 25, 2506-2510.

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SYPRO Ruby Protein Gel Stain binds primarily to proteins through ionic charges of the dye, with basic side chains (lysine, arginine, histidine and to a lesser extent with tyrosine and tryptophan. The fixative solution and SYPRO Ruby stain solution both have an acidic pH, and SYPRO Ruby dye binding increases with protonated basic residues. SYPRO Ruby dye will also bind SDS bound to the proteins and in the gel matrix. The high 50% methanol concentration in the fixative solution is better at stripping out the SDS from the gel matrix, lowering the background staining and allowing for an optimal signal to noise.

Here is a reference on amino acid specificity of SYPRO Ruby Protein Gel Stain:
Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain. Steinberg TH, Chernokalskaya E, Berggren K, Lopez MF, Diwu Z, Haugland RP, Patton WF. Electrophoresis 2006, 21, 486-496.

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For Pro-Q Diamond Phosphoprotein Gel Stain to work properly, it is necessary to delipidate and desalt the sample prior to electrophoresis by following the chloroform/methanol precipitation procedure in the protocol. The Pro-Q Diamond dye will also bind phospholipids and the dye charge interaction with phosphates can be masked by the presence of counter ions and a high salt concentration.

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Yes, Imperial Protein Stain (Cat. Nos. 24615 (1L) and 24617 (3 x 1L) is a ready-to-use colorimetric stain formulated with Coomassie R-250 dye that delivers consistent nanogram-level detection of proteins in polyacrylamide electrophoresis gels.

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E-Gel EX agarose gels separate DNA faster, offer enhanced sensitivity, and provide added flexibility. The stain within the EX gels is proprietary (not based on SYBR technology), though it has the same spectral properties as SYBR Safe DNA Gel Stain. E-Gel EX agarose gels are especially suited for applications when high sensitivity is critical.

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Yes, both the E-Gel GelCapture Acquisition Software and E-Gel GelQuant Express Analysis Software applications are compatible with Windows 10; however, the E-Gel GelQuant Express Analysis Software will require the purchase of an E-Gel Imager Quantitation USB dongle (Cat. No. 4466610):
https://www.thermofisher.com/order/catalog/product/4466610

Please visit the following link (https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/nucleic-acid-gel-electrophoresis/e-gel-electrophoresis-system/e-gel-imager-system/e-gel-software.html) and follow the instructions to download the software.
Note: Please make sure that you download the correct version of the E-Gel GelCapture Software based on the serial number of your instrument.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

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The GelRed or GelGreen nucleic acid dyes can be used with our DNA Ladders. Nucleic acid binding dyes can affect DNA migration during electrophoresis, therefore we highly recommend post-staining of gels. Please follow the gel staining protocol provided by the manufacturer of nucleic acid gel dyes.

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