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View additional product information for Lumio™ Green Detection Kit - FAQs (LC6090)
42 product FAQs found
Lumio试剂具有疏水性，因此很容易穿过细胞膜。无需通过透化处理来帮助该试剂进入细胞内部。
哺乳动物细胞培养基中的血清蛋白（如BSA，66KD）可能会与Lumio试剂发生交叉反应，从而形成非特异性的条带。吸去细胞培养基并在收获细胞后使用PBS润洗哺乳动物细胞3-4遍，有助于减少BSA的非特异性结合。
我们尚未在细胞中发现用于蛋白检测浓度的Lumio试剂具有任何不良效应。我们也未在使用Lumio Green之后发现细胞形态出现任何不良改变。加入Lumio Red后，我们确实发现细胞发生了某些微小的形态改变，不过这一变化在加入试剂24小时之后恢复。
Lumio染色技术的优势在于可同时兼容体内和体外的蛋白标记操作。在体内标记实验中，向细胞中加入Lumio试剂后即可在荧光显微镜下观察到细胞/蛋白。这一效果与GeneBLAzer检测步骤相似，只是GeneBLAzer是基于扩增报告基因信号的酶促反应。GFP的荧光信号只能在细胞（体内）中检测到，因为需要GFP蛋白的正确折叠。相比GeneBLAzer检测法中的bla蛋白（264个氨基酸，29 kDa）和GFP蛋白（27 kDa）而言，Lumio标签非常之小（6个氨基酸，585Da），因此在很大程度上不会对所融合蛋白的功能造成影响。GFP的劣势在于需要融合一个很大的标签蛋白，而且该检测法并非是基于酶法的报告系统。不同于GeneBLAzer检测法和GFP标签，Lumio-标签蛋白可在Lumio试剂处理细胞裂解液或蛋白之后通过凝胶电泳变得可视化。与Lumio和GFP相比，GeneBLAzer检测法在活体细胞的应用中更为灵敏。GeneBLAzer检测法也不同于Lumio和GFP，这一方法能够实现比率化的读数，因此有助于减少样本间的差异。
以下是可能原因和解决方案：
•凝胶处理不恰当或成像平台不干净。处理凝胶或凝胶成像时，不要用手直接接触凝胶。为将所有背景荧光降至最低，每次进行凝胶成像前都应使用纸巾清洁成像系统。
•蛋白上样量过多。降低蛋白浓度或减少上样体积。
•非特异性结合。为将非特异性结合降至最低，应使用Lumio胶内检测增强剂。大肠杆菌裂解物中的某些蛋白（SlyD，21 kDa）和哺乳细胞培养基中的牛血清白蛋白（BSA，66 kDa），会与Lumio绿色试剂发生交叉反应，产生非特异性结合。移除细胞培养基，收获细胞后使用PBS洗涤哺乳细胞3-4次，从而使BSA的非特异性结合降至最低。
以下是可能原因和解决方案：
•标记不恰当。为获得最佳结果，应正确遵循标记方案。应确保在电泳前将Lumio绿色检测试剂加到样品中。避免Lumio凝胶样品缓冲液（4X）暴露于空气中。为保持缓冲液的活性，应在每次使用后立即将Lumio绿色检测试剂和Lumio增强剂放回-20℃。
•蛋白上样量较低或表达水平较低。应按照使用手册的建议，使用总蛋白染色剂对凝胶进行染色，检测凝胶上的总蛋白含量。至少上样1 pmole的Lumio融合蛋白。应确保Lumio标签是框内融合，并且蛋白表达正常。阳性对照与Lumio载体一起提供，以验证蛋白是否表达。
•凝胶长时间暴露于紫外光。Lumio绿色试剂的荧光染色剂对光敏感，所以应避免凝胶长时间暴露于紫外光。
•凝胶未立即显色或成像不恰当。应在从凝胶盒中取出凝胶后进行显色，并在电泳后立即成像。应按着说明书上的建议，使用紫外透照仪或具有合适滤镜的激光扫描仪进行成像。
提示：如果您使用BenchMark荧光蛋白标准品在相同凝胶上电泳并且能够在凝胶上对标准品条带进行成像，则表示凝胶成像是正确的。
97 Da的分子量改变，是由于增强剂覆盖到了不属于Lumio标签的半胱氨酸上，以防止染料与这些半胱氨酸发生非特异性结合。因此，除了Lumio标签中的4个半胱氨酸，其他所有半胱氨酸都将被修饰并产生分子量的改变。
使用BenchMark荧光蛋白标准品，可直接在SDS-PAGE凝胶上观察分子量范围内的Lumio融合蛋白。使用UV透照仪或激光扫描仪可轻松检测到标准品中的蛋白质，其激发和发射波长与Lumio融合蛋白相同。
Lumio胶内检测增强剂是一种独特的溶液，专门用于减少Lumio绿色检测试剂与内源性蛋白质的非特异性结合。
使用标准（Laemmli）SDS-PAGE样品缓冲液制备的样品，不能使用Lumio绿色检测试剂盒进行检测。
为了使染色后的Lumio标签蛋白条带显色，您将需要：
•配有标准摄影机、CCD（电荷耦合器件）相机或冷却型CCD相机的紫外透照仪（302 nm），并带有溴化乙锭滤光片或SYBR Green滤光片。注意：您也可使用365 nm紫外透照仪，但是需要延长凝胶的曝光时间，因为其灵敏度低于302nm紫外透照仪。
•激光扫描仪，带有属于染料最大激发波长（500 nm）的激光线，以及535 nm长通滤波片或以535nm最大发射波长为中心的带通滤波片。配有合适滤光片的激光扫描仪的检测灵敏度比紫外透照仪更高。
使用Lumio绿色检测试剂检测过的蛋白质可以用于质谱（MS）分析。使用Lumio绿色检测试剂检测后，剪切蛋白质条带/点，使用所选方法或根据核心设备的指示来制备用于质谱分析的样品。Lumio绿色检测实验方案会产生以下蛋白质修饰。应确保在质谱分析期间考虑到这些因素：
•蛋白质中的半胱氨酸被修饰，导致每个半胱氨酸增加97.07 Da。
•Lumio绿色试剂中Lumio标签的总分子量为1060 Da（无EDT的Lumio绿色试剂的分子量为475 Da，Lumio标签的分子量为585 Da）。
Lumio标签很小（6个氨基酸，585 Da）。较小的标签不大可能干扰目标蛋白的结构和生物学活性。
该试剂盒的独特配方专用于快速、灵敏和特异性的检测，可检测到1 pmole的Lumio融合蛋白（如1 pmole的30 kDa蛋白质为30 ng）。信号强度可能会改变，取决于蛋白质本身。信号强度也取决于蛋白质条带所含的蛋白质摩尔量，因为1分子的Lumio绿色试剂只能结合蛋白质上的1个Lumio标签。例如，分子量分别为150 kDa和30 kDa的蛋白质上样量均为150 ng/条带，使用Lumio绿色检测试剂盒染色后，30 kDa条带的染色强度高于150 kDa条带。这是因为，在150 ng/条带的相同上样总质量中，150 kDa条带只有1 pmole，而30 kDa条带有5 pmole。
Lumio绿色检测试剂在保存期间可从无色变为粉色。颜色改变不会影响试剂的功能。
Lumio绿色检测试剂的最大激发波长为500 nm，最大发射波长为535 nm。
Lumio绿色检测试剂盒是一种灵敏的、高特异性试剂盒，可用于在电泳前标记Lumio融合蛋白。该试剂盒可直接在聚丙烯酰胺凝胶上使Lumio融合蛋白立即显色。
在避光条件下，Lumio绿色检测试剂盒的组件可在-20℃稳定保存6个月。
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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