Lumio™ Green Detection Kit - FAQs

The Lumio reagent is hydrophobic and can easily pass through the membrane. There is no need to permeabilize the membrane in order to get this reagent into cells.

Serum proteins such as BSA (66 kDa) from the mammalian cell culture medium may cross-react with the Lumio reagent, producing non-specific bands. Removing the cell culture medium and washing the mammalian cells 3-4 times with PBS after harvesting the cells minimizes the non-specific binding from BSA.

We have not experienced negative effects with Lumio reagents at the concentrations used to detect protein in the cells. We also do not see any change in cell morphology when using Lumio Green. After application of the Lumio Red, we do see some minor morphological changes in the cells that are reversed after 24 hours of application of the reagent.

The advantage of Lumio staining is that one can do both in vivo and in vitro protein labeling. For in vivo labeling, load the cells with the Lumio reagent and then visualize the cells/proteins under a fluorescence microscope. This is similar to the GeneBLAzer detection procedure except that GeneBLAzer detection is based on an enzymatic reaction that amplifies the reporter signal. GFP fluorescence can only be detected within the cell (in vivo) because proper protein folding is needed. The Lumio tag is very small (6 amino acids, 585 Da), in contrast to the bla protein in GeneBLAzer detection (264 amino acids, 29 kDa) and the GFP protein (27 kDa), and therefore most likely will not interfere with the function of the protein it is fused to. GFP has the disadvantage of being a large fusion tag and is not an enzymatic-based reporter system. Unlike GeneBLAzer detection and GFP, a Lumio-tagged protein can be visualized on a gel after treating the cell lysate or protein with the Lumio reagent. Compared to Lumio and GFP, GeneBLAzer detection is a more sensitive detection method for use in live cells. Also unlike Lumio and GFP, the GeneBLAzer detection method allows for ratiometric read-outs and thus eliminates sample-to-sample variation.

Yes, fluorescent protein-expressing cells can be fixed using 4% paraformaldehyde in PBS for 10 min followed by one quick PBS rinse and 3 x 5 min washes with 1 mL PBS.

Yes, all of the fluorescent proteins offered by (EmGFP, YFP, CFP, BFP and Cycle 3 GFP) have been humanized for optimal mammalian expression.

We have not used Lumio reagent for in cell labeling in yeast. However the following reference has information about use of Lumio/FlAsH technology for labeling in yeast: Rice MC et al. (2001) In vitro and in vivo nucleotide exchange directed by chimeric RNA/DNA oligonucleotides in Saccharomyces cerevisiae. Mol. Microbiol. 40:857–868. (Note, the article cites FlAsH reagent, which was renamed Lumio Green).
Other helpful references on use of FlAsH (Lumio) may be found in this review article: Cavagnero S, Jungbauer LM (2005) Painting protein misfolding in the cell in real time with an atomic-scale brush. Trends Biotechnol 23:157-162.

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The FlAsH-EDT2 reagent has been renamed Lumio Green Labeling Reagent. It is available in the Lumio Green In-Cell Labeling Kit, Cat. No. 12589-057. While the kit is designed for staining live cells expressing proteins containing the Lumio fusion tag, you may substitute the Lumio Green labeling reagent directly into your existing protein labeling protocol. The kit contains the Lumio Green reagent and Disperse Blue 3 for background suppression. We also have available the Lumio Red In-Cell Labeling Kit, Cat. No. 12589-040.

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Yes. After Lumio reagent detection, cut out the band or spot and prepare the samples as you normally would for mass spec analysis. Be sure to account for the following protein modifications: (1) The molecular weight of the Lumio tag plus other potential fusion tags (this will vary depending on the expression construct). (2) The molecular weight of the Lumio Green Reagent without EDT is 480 Da.

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Serum proteins (BSA, 66 kDa) from the mammalian cell culture medium may cross-react with the Lumio Reagent producing non-specific bands. Removing the cell culture medium and washing the mammalian cells 3-4 times with PBS after harvesting the cells minimizes the non-specific binding from BSA. The BSA band may show up as a 66 kDa band.

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Here are possible causes and solutions:

- Improper handling of gels or dirty imaging platform. Avoid touching the gel with bare hands while handling or imaging the gel. Always clean the imaging platform with a paper towel prior to imaging the gel to minimize any background fluorescence.
- Protein was overloaded. Decrease the protein concentration or lower the sample volume.
- Non-specific bands. Use the Lumio In-Gel Detection Enhancer to minimize non-specific binding. Certain proteins from E. coli lysates (SlyD, 21 kDa) and serum proteins (BSA, 66 kDa) from the mammalian cell culture medium may cross-react with the Lumio Green Reagent producing non-specific bands. Removing the cell culture medium and washing the mammalian cells 3-4 times with PBS after harvesting the cells minimizes the non-specific binding from BSA.

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Here are possible causes and solutions:

- Improper labeling. Make sure that the labeling protocol is correctly followed to obtain the best results. Make sure you have added the Lumio Green Detection Reagent to the samples prior to electrophoresis. Limit exposure of the Lumio Gel Sample Buffer (4X) to air. Always return the Lumio Green Reagent and Lumio Enhancer to ?20 degrees C immediately after use to preserve the activity of buffers.
- Low protein load or low expression level. Check total protein loaded on the gel by staining the gel with a total protein stain as described in the manual. Load at least 1 pmole of the Lumio fusion protein. Make sure the Lumio tag is in frame and the protein is expressed properly. A positive control is supplied with the Lumio vectors to verify the expression protocol.
- The gel is exposed to UV light for a long time. The fluorescent dye of the Lumio Green Reagent is sensitive to photobleaching, so avoid exposing the gel to UV light for a long time.
- The gel is not visualized immediately or imaged properly. Be sure to visualize the gel after removing the gel from the cassette and view the gel immediately after electrophoresis. Use a UV transilluminator or a laser-based scanner using appropriate filters as described in the manual. Tip: If you have run BenchMark Fluorescent Protein Standard on the same gel and can view the standard bands on the gel, then you are imaging the gel properly.

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The 97 Da molecular weight change is due to the Enhancer capping the cysteines that aren't part of the Lumio tag to prevent non-specific binding of the dye to the cysteines. So all cysteines except the 4 in the Lumio tag will be modified and show this change in molecular weight.

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The BenchMark Fluorescent Protein Standard allows direct visualization of molecular weight ranges of Lumio fusion proteins on an SDS-PAGE gel. The proteins in the standard are easily detected using a UV transilluminator or a laser-based scanner at the same excitation and emission wavelengths as your Lumio fusion protein.

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The Lumio In-Gel Detection Enhancer is a proprietary solution and is designed to reduce the non-specific binding of Lumio Green Detection Reagent with endogenous proteins.

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Samples prepared in standard (Laemmli) SDS-PAGE sample buffer are not compatible for use with the Lumio Green Detection Kit.

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To visualize Lumio-tagged protein bands after staining, you will need one of the following:

- UV transilluminator (302 nm) equipped with a standard video camera, CCD (Charged Couple Device) camera, or a cooled CCD camera with ethidium bromide filter or SYBR Green filter. Note: You can use a 365 nm UV transilluminator, but you may have to expose the gel for a longer time, as the sensitivity is lower than using 302 nm UV transillumination.
- Laser-based scanner with a laser line that falls within the excitation maxima of the stain (500 nm), and a 535 nm long pass filter or a band pass filter centered near the emission maxima of 535 nm. The sensitivity of detection is higher with laser-based scanners equipped with appropriate filters than with UV transillumination.

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Proteins detected with the Lumio Green Detection Kit are compatible with mass spectrometry (MS) analysis. After detection with Lumio Green Detection Kit, excise the protein band/spot and prepare the samples for MS analysis using a method of choice or as directed by your core facility. The Lumio Green Detection protocol produces the following protein modifications. Be sure to account for these during MS analysis:

- Cysteines in the protein are modified and will result in the addition of 97.07 Da to each cysteine.
- The total molecular weight of the Lumio tag with the Lumio Green Reagent is 1060 Da (molecular weight of the Lumio Green Reagent without EDT is 475 Da and molecular weight of the Lumio tag is 585 Da).

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The Lumio tag is small (6 amino acids, 585 Da). The small size of the tag is unlikely to interfere with the structure or biological activity of the protein of interest.

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The kit is specially formulated for fast, sensitive, and specific detection, and is capable of detecting 1 pmole of a Lumio fusion protein (e.g., 1 pmole of a 30 kDa protein is 30 ng). The signal intensity may vary and is dependent on the individual protein. The signal intensity is also dependent on the number of moles of protein contained in a protein band because 1 molecule of Lumio Green Reagent binds to only 1 Lumio tag on the protein. For example, if you load 150 ng/band of 2 proteins with molecular weights of 150 kDa and 30 kDa, respectively, after detection with Lumio Green Detection Kit, the 30 kDa band fluoresces more intensely than the 150 kDa band. This is because there is only 1 pmole of the 150 kDa band while there are 5 pmoles of the 30 kDa band in the total mass loaded (150 ng/band).

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The color of the Lumio Green Detection Reagent may change from colorless to pink during storage. This color change does not affect the functioning of the reagent.

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The maximum excitation wavelength for Lumio Green Detection Reagent is at 500 nm and maximum emission wavelength is at 535 nm.

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The Lumio Green Detection kit is a sensitive and highly specific kit for labeling Lumio fusion proteins prior to electrophoresis. It enables immediate visualization of Lumio fusion protein bands directly in a polyacrylamide gel.

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We recommend storing the Lumio Green Detection kit components at -20 degrees C, protected from light, where it is stable for one year from date of shipment.

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