SYPRO™ Ruby Protein Gel Stain, 5 L - FAQs

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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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.

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SYPRO Ruby Protein Gel Stain is not stable beyond about a year. The dye begins to precipitate out from solution (self-aggregate) over time and will show a lower staining intensity of protein bands and increased ‘debris' or ‘speckles' on the surface of the gel. It is not possible to filter the stain to remove dye precipitate, as the dye sticks to most paper and membrane filters and will be removed from the staining solution.

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Shadowing around the bands means that the gel background staining of SDS is too high. Destain the gel in 10% methanol/7% acetic acid a little longer, approximately another 30 minutes and then give it a good water wash. In the future, try fixing the gel longer, at least another 30 minutes, to better remove SDS from the gel and reduce initial background staining.

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The gel would need to be sequentially stained and imaged after each stain. The order of staining would be: InVision His-Tag In-Gel Stain (Cat. No. LC6030) then Image then Pro-Q Diamond Phosphoprotein Gel Stain then Image then Pro-Q Emerald 300 or 488 Glycoprotein Gel Stain then SYPRO Ruby Protein Gel Stain then Coomassie Blue or Silver Stain then Image

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Yes. We recommend staining with SYPRO Ruby Blot Stain after staining with Pro-Q Diamond Phosphoprotein Blot Stain.

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No. SYPRO Ruby Protein Blot Stain is not compatible with cationic membranes, such as Immobilon CD or Immobilon N membranes. Since it is an anionic dye, it binds non-specifically to the membrane. This is also true for amido black, Coomassie Blue, ponceau red and just about any other total protein blot stain. The Immobilon P or Immobilon FL membranes are recommended for use with SYPRO Ruby Protein Blot Stain.

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SYPRO Ruby stain is actually brighter when dry, so a dry blot is better for epiillumination. If you only have a transilluminator, blots are more transparent when wet, so wet illumination will give a brighter signal and lower background.

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Yes. SYPRO Ruby stain is compatible with subsequent western detection using colorimetric, fluorogenic, and chemiluminescent detection techniques including BCIP/NBT, ECL, and CDP-Star reagent. For best western detection sensitivity, we recommend destaining the blot in 20% methanol, 150 mM Tris, pH 8.8 for 10 min followed by four 1 min rinses in deionized water to remove most of the SYPRO Ruby dye before performing the western detection procedure.

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Dried blots stained with SYPRO Ruby blot stain are very photostable and can be stored for long periods of time at room temperature, in the dark, including taping into a notebook.

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Yes, staining a dry PVDF membrane (face staining) gives a lower non-specific background signal and results in a better signal to noise compared to a blot that has been re-wet in methanol (immersion staining). Background is also lower because the back side of the membrane does not see stain. Areas of the blot that are not completely dry or have a lot of residual SDS will wet out and show up as a darker stained background. If this is a problem, the entire PVDF blot can be re-wet in 100% methanol, so that the entire background is stained the same. Both the stained protein signal and blot background will be brighter. You should also increase the number of water washes or time for immersion stained blots. Drying the blot also completely binds the proteins to the membrane.

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Yes. Blots can be left in water indefinitely without destaining the stained proteins. Longer water washes can help to reduce high non-specific background, especially for immersion-stained PVDF membranes.

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Yes, the 15 min incubation time is a minimum time. The blot will not overstain if left in SYPRO Ruby Blot Stain longer than 15 min.

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No, not completely. You can incubate the gel or PVDF blot in several changes of 20-50% methanol, 150 mM Tris, pH 9 to remove the majority of the dye, but it will not be completely gone.

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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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With the Safe Imager 2.0 Blue-Light Transilluminator, it takes 8-10 minutes of constant ultraviolet illumination to cause excessive photobleaching. The bleaching rate will vary with the intensity of your light source, but under most conditions this probably will not be a problem. Even when bleached, gels can be prestained with SYPRO stains with only a small decrease in sensitivity.

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SYPRO Ruby Gel Stain has a very slow off-rate. In fact, it is very difficult to completely strip the dye out of the gel once it has bound proteins. Stained gels can be stored at 4 degrees C, protected from light, for at least several months with little loss in signal. Just keep hydrated in a little water with 2-5 mM sodium azide as a preservative or in 7% acetic acid. For more permanent storage, dry the gel or vacuum seal in 1-5 mL of SYPRO Ruby stain containing 2-5 mM sodium azide and store at 4 degrees C. Seal-A-Meal food storage bags are useful for this method of preservation.

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SYPRO Ruby Gel Stain is an endpoint stain, which means that once it has reached saturation binding of proteins, it will no longer continue to stain proteins or increase background signal, unlike silver stains. Gels can be left in SYPRO Ruby Gel Stain for months (4 degrees C recommended) without over-staining.

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No, the SYPRO Ruby Protein Gel Stain and Blot Stain have very different formulations that are optimized for their respective usage and are not interchangeable. Blots or gels stained with the alternate SYPRO Ruby product will have suboptimal staining intensities and high backgrounds.

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Yes, SYPRO Ruby stained gels and blots can be stained with any Coomassie Blue dye-based stain and will yield similar results as a gel or blot stained only with Coomassie Blue dye. The fluorescence of SYPRO Ruby will be lost after Coomassie Blue staining.

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SYPRO Ruby stained gels can be post-stained with any silver stain, such as SilverXpress Silver Staining Kit (Cat. No. LC6100) and SilverQuest Silver Staining Kit (Cat. No. LC6070), and double staining is actually an excellent method to enhance the sensitivity obtained with individually stained SYPRO Ruby or silver stained gels. Simply follow the normal 10% methanol, 7% acetic acid destain after SYPRO Ruby staining, wash in water, if desired, to image the SYPRO Ruby signal and then perform the silver stain procedure. It is not necessary to perform the fixation step in the silver stain protocol, as the gels have already been fixed. The silver ions are apparently attracted to the SYPRO Ruby dye, enhancing the silver deposition around the proteins and thus the signal. Once a SYPRO Ruby stained gel has been silver stained, the SYPRO Ruby fluorescence signal can no longer be detected.

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We have only tested proteins as small as 6,000 Da, but if smaller proteins can be resolved in the gel and contain lysine, arginine or histidine basic amino acid residues, then it is likely that they will be detected too.

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The SYPRO Ruby stain does not interfere with mass spectrometry. After staining with SYPRO Ruby stain, the protein band or spot can be trypsinized and sent for mass spectrometry analysis. Although the SYPRO Ruby stain does not interfere with mass spectrometry, occasionally we see some peaks that are due to the SYPRO Ruby dye. These are a symmetrical grouping of peaks centering around 1257 and 1279 MW.

Reference: Parker K, Garrels J, Hines W, Butler E, McKee A, Patterson D, Martin S. Electrophoresis 1998, 19, 1920-1932.

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SYPRO Ruby dye (as well as SYPRO Orange, Coomassie Fluor Orange and the nucleic acid stains SYBR Safe, SYBR Gold, and SYBR Green I and II) fluoresces nicely on the Safe Imager 2.0 Blue-Light Transilluminator (Cat. No. G6600) or other similar blue light transilluminators with excitation near 470 nm. The gel can be viewed with amber glasses that are supplied with the unit. UV light transilluminators equipped with 302 nm or 365 nm bulbs can be used as well, but would require UV-protective equipment during use.

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For many routine gel or blot images that do not require analysis, a simple digital camera or camera phone, such as an iphone camera, can be used in combination with a UV or blue light transilluminator, such as the Safe Imager 2.0 Blue-Light Transilluminator (Cat. No. G6600). Place the gel on the surface of the transilluminator and cover with the amber plastic filter. Take a picture through the amber plastic to reduce background illumination and improve sensitivity. Blots are best imaged from above the membrane (epiillumination) rather than through the membrane (transillumination). Place the light box on its side with the blot face-up on the table. Hold the amber plastic filter up close to the camera lens to take a picture.

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A handy list of instruments that work well with SYPRO Ruby stain is available here (https://tools.thermofisher.com/content/sfs/manuals/x11791.pdf).

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Yes, the best place to stop for the day is during the first fixation step. Gels can be left overnight up to several days in the first fixation solution with no effect on the resulting staining. One long fixation is sufficient to remove SDS from the gel, so it is not necessary to repeat the fixation step, thus reducing solvent usage. An overnight or longer fixation will dehydrate the gel matrix significantly, so that it will be reduced in size and turn opaque white. You must rehydrate the gel before microwaving. Simply rock for about 5 min in water or SYPRO Ruby Gel Stain to rehydrate the gel. Check to make sure that the gel is floating in solution and has not stuck to the bottom of the staining dish before microwaving, or it will rehydrate unevenly and stain unevenly. After the gel has rehydrated back to its original size, it can then be microwaved in the SYPRO Ruby Gel Stain.

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No, 23 min happens to be the time that is left over from the total 30 min stain time after subtracting the microwave and 5 min incubation times. The reality is that those are the minimum times used so that the whole fix, stain, destain procedure can be complete in 90 min. The exact timing of the microwave staining step 2.2 does not need to be followed stringently. Gels can be stained longer than 30 min, but background will also gradually increase along with the protein signal, so that sensitivity is not improved by staining longer than 30 min using the microwave protocol. Generally, staining for 30 to 90 min will give similar results. The maximum end-point signal intensity is reached after about 5 h, the same as that achieved using the overnight staining protocol.

Gels just need to be microwaved twice during the 30 min stain time to near boiling, so that only small bubbles are formed. Over-boiling can bump the staining solution, and in some cases, scorch and damage the gel.

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No, other commonly used gel fixatives can be used, including reducing the methanol concentration to 10% methanol in 7% acetic acid. SYPRO Ruby stain itself will fix proteins in the gel and there is no need for a separate fixation step to stain proteins with reasonably good results. The 50% methanol/7% acetic acid fixation recommended in the protocol has been determined to best remove residual SDS from the gel matrix and thus give the lowest background and optimal sensitivity compared to other fixation methods. Reduced methanol concentrations could result in a heavily stained SDS front at the bottom of the gel, which will reduce detection sensitivity for low molecular weight proteins running near this region.

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Yes, but ethanol/acetic acid will produce ethyl acetate, which has a strong odor, so you should do this fixation in a fume hood.

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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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Proteins stained with SYPRO Ruby Protein Gel Stain, or any gel stain where the proteins are fixed in the gel, cannot be blotted onto membranes. Both the fixation step and the low pH of the SYPRO Ruby Gel Stain solution precipitate proteins into the gel matrix to prevent diffusion during staining, and thus also efficient transfer onto membranes. We recommend using SYPRO Tangerine Protein Gel Stain, which is a neutral pH, simple saline solution-based, non-fixative gel stain to prestain proteins before transfer.

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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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If a protein has a low content of basic residues, you will see a lower level of staining relative to a protein with a higher content of basic residues. Very heavily glycosylated proteins are an example of proteins with low content of basic residues and may not stain as well as non-glycosylated or lightly glycosylated proteins, thus lowering detection sensitivity. If a protein does not have any basic residues, as can exist for synthetic constructs, it will not bind the dye.

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Fluorescence intensity varies linearly with protein quantity over nearly three orders of magnitude, from 1 ng to 1000 ng. SYPRO Ruby stain has a broader linear range than silver staining, basically, the more protein you have, the more SYPRO Ruby stain that binds.

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Since SYPRO Ruby Protein Gel Stain binds primarily to basic protein residues instead of SDS, it can be used as an endpoint stain (direct, saturation binding with a low off-rate) whose performance is not affected by deviations in fixation, staining or destaining solution volumes or incubation times, but just the amount of protein that is present. The strong 50% methanol/7% acetic acid fixation in the SYPRO Ruby stain protocol removes most SDS bound to proteins and in the gel matrix, so that the signal is due to direct protein binding. Stains that bind protein indirectly via SDS/protein association (such as Deep Purple, Lucy, SYPRO Orange/Red/Tangerine, Nile Red dyes) show higher protein to protein and gel to gel variation depending on how much SDS is bound to the protein and present in the gel matrix. Proteins that do not bind SDS to saturation, such as heavily glycosylated or phosphorylated proteins will show reduced staining with indirect SDS binding protein stains.

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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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We recommend leaving the gel in the stain overnight. There is no need to add the extra salt solution as recommended in the manual because there are no buffer salts and SDS to worry about, and that would end up inhibiting the staining too much.

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