Search
Search
查看更多产品信息 NT-GFP Fusion TOPO™ Expression Kit - FAQs (K481001)
37 个常见问题解答
pBlue-TOPO中的lacZ报告基因上游隐含了一个原核启动子，通过X-Gal平板进行筛选时，大肠杆菌的转化子会呈现浅蓝色。因此，我们不推荐用户使用pBlue-TOPO来评估大肠杆菌中的启动子功能。不过，pGlow-TOPO适用于此类研究。请注意pBlue-TOPO载体中β-半乳糖苷酶的背景表达不会出现于哺乳动物细胞中。
是的，我们提供pBlue-TOPO和pGlow-TOPO载体，目的基因的DNA序列能够轻松插入到β-半乳糖苷酶或Cycle 3 GFP基因上游。
pBlue-TOPO是进行低转录活性启动子功能分析的理想载体，，因为β-半乳糖苷酶检测实验易于操作，且可以在非常低的表达水平下进行定量。pGlow-TOPO是在天然活体细胞中针对启动子元件开展非侵入性分析的理想之选。在使用带有野生型GFP滤光片组的显微镜，或荧光激活的细胞分选法的过程中，Cycle 3 GFP的荧光性质能够满足几乎任何细胞类型或物种的体内检测需求。
我们推荐在进行β-半乳糖苷酶染色前观察GFP荧光。这是因为β-半乳糖苷酶染色过程会产生非常高的自发荧光，从而干扰GFP荧光的检测。
Cycle 3 GFP的荧光可通过适用于野生型GFP的滤光片组来完成检测（因为它们具有相同的荧光光谱）。在内部实验中，我们一般使用Omega Optical公司所提供的XF76滤光片组。Cycle 3 GFP的激发光谱为395 nm，发射光谱为507 nm。您也可对发射光谱进行观察，并记录200-800 nm处的发射情况。
Cycle 3 GFP的荧光可通过具有适当滤光片和截止波长的任意种类的荧光分析仪器进行定量。在内部实验中，我们一般使用Hitachi F-2000荧光分光光度计。我们应用此仪器的常规方案如下：
使用PBS稀释样本（也可使用Tris缓冲液或水）。裂解液的用量需要基于GFP的浓度来进行调整。这一用量一般是根据经验来确定的。第一个需要考虑的是样本浓度应处于荧光分析仪器的线性区间。我们在一个比色皿（已添加1ml PBS）中应使用5-50 µL的样本量。如需对这些读值进行分析比较，则最好能够将裂解液的用量按照转染效率和总蛋白浓度进行归一化运算，以帮助获得最为稳定的结果。
EmGFP，YFP，CFP和BFP均可通过标准的FITC滤光片组和参数设定来进行检测。不过，如需获得荧光信号的最佳检测结果，我们推荐用户针对每一种荧光蛋白的激发与发射波长范围来优化滤光片组。推荐的滤光片组如下所述：
EmGFP：Omega滤光片组XF100
YFP:Omega滤光片组XF1042
Chroma滤光片组41028
CFP:Omega滤光片组XF114
Chroma滤光片组31044
BFP:Omega滤光片组XF10
Chroma滤光片组31021
如需索取滤光片组的相关信息，请直接联系Omega Optical公司（www.omegafilters.com）或Chroma 技术公司（www.chroma.com）。
我们所提供荧光蛋白的激发/发射最大波长如下所述：
EmGFP：激发：487 nm；发射：509 nm
YFP：激发：514 nm；发射：527 nm
BFP：激发：308-383 nm；发射：440-447 nm
CFP：激发：452 nm；发射：505 nm
Cycle 3 GFP：第一激发波长：395 nm；第二激发波长：478 nm；发射：507 nm
是的，我们所提供的所有荧光蛋白（EmGFP，YFP，CFP，BFP和Cycle 3 GFP）均经过人源基因密码子优化以适合哺乳动物表达系统。
EmGFP是EGFP的下一代变体，在哺乳动物体内表达效果更优。EmGFP和EGFP均可通过相同的滤光片组（FITC）和参数设定来实现可视化操作。在使用了推荐的滤光片组和参数设定的条件下，Cycle 3 GFP与EGFP或EmGFP一样明亮。不过，当使用FITC滤光片组及相关设定时，Cycle 3 GFP不如EmGFP或EGFP明亮。
EGFP的激发/发射最大波长：488 nm/507–509 nm
EmGFP的激发/发射最大波长：487 nm/509 nm
Cycle 3 GFP的激发/发射最大波长：395 nm （第一）和478 nm（第二）/507 nm
除了进行了关键突变以增加Clontech荧光蛋白亮度之外，我们还进行了遗传增强元件改造，以提升荧光量子产率。平行对比结果显示Vivid Colors荧光蛋白表达载体的荧光强度至少与对应的Clontech BD Living Colors荧光蛋白表达载体相当（或更好）。
这里列举了一些可能的原因与解决方案：
•所用检测法可能不适当或不够灵敏： ◦我们推荐您优化检测方案或寻找更为灵敏的方法。如果使用考马斯亮蓝染色/银染法检测过该蛋白，我们则推荐您使用免疫印迹法来增加检测灵敏度。裂解产物中存在的内源蛋白可能会在考马斯亮蓝染色/银染过程中掩盖目的蛋白。如果可能，我们推荐您在免疫印迹实验中包括一个阳性对照。
•筛选到的克隆数不够：至少筛选出20个克隆。
•在稳转筛选中使用了不适当的抗生素浓度：请确保正确获取了抗生素的杀死曲线。由于某一既定抗生素的效力依赖于细胞类型，血清，培养基和培养技术，因此必须在每次进行稳定筛选的时候确定抗生素的用量。如果采用的培养基或血清条件明显不同，则即使是我们所提供的稳转细胞系对于我们推荐的剂量也可能出现更敏感或更不敏感的情况。
•基因产物（即使低水平）的表达可能与该细胞系的生长不相容（如毒性基因）：使用一个可诱导的表达系统。
•阴性克隆可能由基因表达的关键载体位点处优先发生了线性化所致：在一个不影响表达的位点实施载体线性化，如在细菌抗药性标志物序列中。
这里列举了一些可能的原因与解决方案：
•尝试试剂盒自带的表达对照。
•可能的检测问题：
◦检测瞬转的表达蛋白可能有难度，因为转染效率可能过低，以致用于整个转染群体的评估手段无法成功实现检测。我们推荐您通过稳转筛选或采用能够逐个检测单一细胞的技术手段来优化您的转染操作。您也可尝试通过改变启动子或细胞类型来提高表达水平。
◦细胞中的蛋白表达水平对于所选择的检测方法来说可能过低。我们推荐您优化检测方案或寻找更为灵敏的方法。如果使用考马斯亮蓝染色/银染法检测过该蛋白，我们则推荐您使用免疫印迹法来增加检测灵敏度。裂解产物中存在的内源蛋白可能会在考马斯亮蓝染色/银染过程中掩盖目的蛋白。如果可能，我们推荐您在免疫印迹实验中包括一个阳性对照。
◾蛋白可能降解或截短了：使用Northern杂交进行检测。
◾可能的时程问题：由于蛋白表达随时间延长而发生的变化依赖该蛋白的天然属性，我们一般推荐您先获取一份表达的时程曲线。尝试进行一次时程分析将帮助您确定最优的表达时间窗。
◾可能的克隆问题：通过限制性酶切和/或测序来验证克隆。
不可以；新霉素对哺乳动物细胞有毒性。我们推荐您使用Geneticin（又称 G418硫酸盐)，这一产品的毒性较低，是在哺乳动物细胞中进行有效筛选的新霉素的替代品。
即使缺乏Kozak序列，翻译也还是会在核糖体遇到的第一个ATG处启始，不过启始效率可能相对较低。只要处于最初ATG的阅读框内，任何下游的插入序列都可能表达为融合蛋白，不过如果这里没有Kozak保守序列，则蛋白的表达水平预期会比较低。如果载体中包含一个非Kozak型的保守ATG，我们则推荐您将基因克隆至该ATG上游，再包含一个Kozak序列来优化表达效果。
我们提供pJTI R4 Exp CMV EmGFP pA载体，货号A14146，您可使用这一产品来监控转染和表达情况。
在小鼠细胞系中，人们已知CMV启动子的效率会随时间延长而逐渐下降。因此，我们推荐您使用一款非CMV型的载体，如EF1α或UbC启动子，以在小鼠细胞系中长时间表达蛋白。
保守的Kozak序列为A/G NNATGG，其中的ATG表示起始密码子。ATG周围的核苷酸点突变会影响翻译效率。尽管我们通常情况下都推荐加入一段Kozak保守序列，不过这一操作的必要性还是基于具体的目的基因，一般只需ATG就足以高效地启始翻译过程。最佳的建议是保持cDNA中天然起始位点，除非确定这一位点的功能性不理想。如果从表达的角度来考虑，推荐构建并测试两种载体，一个具有天然的起始位点，另一个具有保守的Kozak序列。通常情况下，所有具有N-融合表达的表达载体都已经包含了一个翻译起始位点。
ATG通常对于高效的翻译启始是足够的，尽管翻译效率要视目的基因而定。最佳的建议应是保持cDNA中天然起始位点，除非确定这一位点的功能性不理想。如果从表达的角度来考虑，推荐构建并测试两种载体，一个具有天然的起始位点，另一个具有保守的Kozak序列。通常情况下，所有N-端融合型表达载体都已包含了一个RBS或翻译起始位点。
原核生物mRNA含有Shine-Dalgarno序列，也称为核糖体结合位点(RBS)，它是由AUG起始密码子5’端的多嘌呤序列AGGAGG组成。该序列与16S rRNA 3’端的互补，有助于mRNA有效结合到核糖体上。同理，真核生物（特别是哺乳动物）mRNA也含有完成有效翻译所需的重要序列信息。然而，Kozak序列不是真正的核糖体结合位点，而是一种翻译起始增强子。Kozak共有序列是ACCAUGG，其中AUG是起始密码子。-3位的嘌呤(A/G)具有重要作用；若-3位是一个嘧啶(C/T)，翻译过程会对-1、-2和+4位的改变更敏感。当-3位从嘌呤变为嘧啶时，可使表达水平降低多达95%。+4位对表达水平的影响相对较小，可以使表达水平降低约50%。
注：果蝇的最佳Kozak序列稍有不同，酵母完全不遵循这些规则。见下列参考文献：
•Foreign Gene Expression in Yeast: a Review. Yeast, vol. 8, p. 423-488 (1992).
•Caveneer, Nucleic Acids Research, vol. 15, no. 4, p. 1353-1361 (1987).
pBlue-TOPO contains a cryptic prokaryotic promoter upstream of the lacZ reporter gene, due to which E. coli transformants may appear to be light blue when screened on plates containing X-Gal. Hence, we do not recommend using pBlue-TOPO to evaluate promoter function in E. coli. However, pGlow-TOPO can be used for these studies. Note that background expression of beta-galactosidase from pBlue-TOPO does not occur in mammalian cells.
Yes, we do offer the pBlue-TOPO and pGlow-TOPO vectors that facilitate cloning of the DNA sequence of interest directly upstream of either the b-galactosidase or Cycle 3 GFP gene, respectively.
pBlue-TOPO is ideal for functional analysis of promoters with low transcriptional activity, since assays for beta-galactosidase are easy to perform and are quantitative at very low levels of expression. pGlow-TOPO is ideal for non-invasive analysis of promoter elements within intact, living cells. The fluorescent property of Cycle 3 GFP allows in vivo detection in virtually any cell type or species using microscopy with wild-type GFP filter sets or by fluorescence-activated cell sorting methods.
We recommend looking for GFP fluorescence before staining for beta-galactosidase. This is because the beta-galactosidase staining process produces a very high autofluorescence that will interfere with detection of GFP fluorescence.
Cycle 3 GFP fluorescence can be detected using a filter set designed to detect wild-type GFP (since they have the same fluorescence spectra). In-house, we use the XF76 filter set from Omega Optical. For Cycle 3 GFP, excite at 395 nm and read emission at 507 nm. You can also look at the emission spectra and record emissions from 200-800 nm.
Cycle 3 GFP fluorescence can be quantitated with any type of fluorometer with the appropriate filters and cut-off wavelengths. In-house, we have a Hitachi F-2000 Fluorescence Spectrophotometer. Our general protocol using this machine is as follows:
Dilute samples in PBS (although Tris or water would be okay). The amount of lysate to be used will of course depend upon the concentration of GFP. This will have to be determined empirically. The primary consideration is that one needs to be in the linear range of the fluorometer. We have used quantities from 5-50 µL in 1 mL of PBS in a cuvette. If readings are going to be internally compared, the most consistent results will be obtained if the amounts of lysate used are normalized to either the transfection efficiency or the total protein concentration.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
EmGFP, YFP, CFP, and BFP can be detected using standard FITC filter sets and settings. However, for optimal detection of the fluorescence signal, filter sets optimized for detection within the excitation and emission ranges for each fluorescent protein are recommended. The recommended filter sets are as follows: EmGFP: Omega filter set XF100 YFP: Omega filter set XF1042 Chroma filter set 41028 CFP: Omega filter set XF114 Chroma filter set 31044 BFP: Omega filter set XF10 Chroma filter set 31021 For information on obtaining filter sets, please contact Omega Optical, Inc. (www.omegafilters.com) or Chroma Technology Corporation (www.chroma.com) directly.
Excitation and emission maxima for our fluorescent proteins are as follows:
- EmGFP: Excitation: 487 nm; Emission: 509 nm
- YFP: Excitation: 514 nm; Emission: 527 nm
- BFP: Excitation: 308-383 nm; Emission: 440-447 nm
- CFP: Excitation: 452 nm; Emission: 505 nm
- Cycle 3 GFP: Primary excitation: 395 nm; Secondary Excitation: 478 nm; Emission: 507 nm
Yes, all of the fluorescent proteins offered by us (EmGFP, YFP, CFP, BFP, and Cycle 3 GFP) have been humanized for optimal mammalian expression.
EmGFP is the next-generation variant of EGFP, and it has been further optimized for mammalian expression. Both EmGFP and EGFP can be visualized using the same filter sets (FITC) and settings. When used with the recommended filter sets and settings, Cycle 3 GFP is as bright as EGFP or EmGFP. However, when used with FITC filter sets and settings, Cycle 3 GFP is not as bright as EmGFP or EGFP.
- Excitation/emission maxima for EGFP: 488 nm/507-509 nm
- Excitation/emission maxima for EmGFP: 487 nm/509 nm
- Excitation/emission maxima for Cycle 3 GFP: 395 nm (primary) and 478 nm (secondary)/507 nm
In addition to the key mutations that enhanced the brightness of the Clontech fluorescent proteins, we have added further genetic enhancements to the fluorescent proteins to increase the quantum yield. Side-by-side comparisons have shown the fluorescence intensity of our Vivid Colors fluorescent protein expression vectors to be at least equivalent (or better than) the comparable Clontech BD Living Colors fluorescent protein expression vectors.
Here are possible causes and solutions:
Detection method may not be appropriate or sensitive enough:
- We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot.
- Insufficient number of clones screened: Screen at least 20 clones.
- Inappropriate antibiotic concentration used for stable selection: Make sure the antibiotic kill curve was performed correctly. Since the potency of a given antibiotic depends upon cell type, serum, medium, and culture technique, the dose must be determined each time a stable selection is performed. Even the stable cell lines we offer may be more or less sensitive to the dose we recommend if the medium or serum is significantly different.
- Expression of gene product (even low level) may not be compatible with growth of the cell line: Use an inducible expression system.
- Negative clones may result from preferential linearization at a vector site critical for expression of the gene of interest: Linearize the vector at a site that is not critical for expression, such as within the bacterial resistance marker.
Here are possible causes and solutions:
- Try the control expression that is included in the kit
Possible detection problem:
- Detection of expressed protein may not be possible in a transient transfection, since the transfection efficiency may be too low for detection by methods that assess the entire transfected population. We recommend optimizing the transfection efficiency, doing stable selection, or using methods that permit examination of individual cells. You can also increase the level of expression by changing the promoter or cell type.
- Expression within the cell may be too low for the chosen detection method. We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot.
Protein might be degraded or truncated: Check on a Northern.
Possible time-course issue: Since the expression of a protein over time will depend upon the nature of the protein, we always recommend doing a time course for expression. A pilot time-course assay will help to determine the optimal window for expression.
Possible cloning issues: Verify clones by restriction digestion and/or sequencing.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
No; neomycin is toxic to mammalian cells. We recommend using Geneticin (a.k.a. G418 Sulfate), as it is a less toxic and very effective alternative for selection in mammalian cells.
Translation initiation will occur at the first ATG encountered by the ribosome, although in the absence of a Kozak sequence, initiation will be relatively weak. Any insert downstream would express a fusion protein if it is in frame with this initial ATG, but levels of expressed protein are predicted to be low if there is a non-Kozak consensus sequence. If the vector contains a non-Kozak consensus ATG, we recommend that you clone your gene upstream of that ATG and include a Kozak sequence for optimal expression.
We offer pJTI R4 Exp CMV EmGFP pA Vector, Cat. No. A14146, which you can use to monitor your transfection and expression.
The CMV promoter is known to be downregulated over time in mouse cell lines. Hence, we recommend using one of our non-CMV vectors, such as those with the EF1alpha or UbC promoter, for long-term expression in mouse cell lines.
The consensus Kozak sequence is A/G NNATGG, where the ATG indicates the initiation codon. Point mutations in the nucleotides surrounding the ATG have been shown to modulate translation efficiency. Although we make a general recommendation to include a Kozak consensus sequence, the necessity depends on the gene of interest and often, the ATG alone may be sufficient for efficient translation initiation. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a consensus Kozak. In general, all expression vectors that have an N-terminal fusion will already have an initiation site for translation.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Eukaryotic (and specifically mammalian) mRNA contains sequence information that is important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.