Invitrogen™ NT-GFP Fusion TOPO™ Expression Kit

Catalog No.	Quantity
K481001	20 reactions

Catalog No. K481001 Supplier Invitrogen™ Supplier No. K481001

Description

Includes

GFP Fusion TOPO Expression Kits contain two boxes. The GFP Fusion TOPO TA Cloning box contains linearized and topoisomerase I-activated cloning vector, dNTPs, salt solution control PCR template and primers, forward and reverse primers for sequencing and PCR screening, and a GFP expression control plasmid. The One Shot™ box contains transformation reagents including single-use, 50μL aliquots of TOP10 Chemically Competent E. coli, S.O.C. medium, and supercoiled control plasmid.

Designed to allow fusion of protein of interest to Cycle 3 GFP protein

• Provide topoisomerase-I activated pcDNA3.1/NT-GFP-TOPO vector
• Taq-amplified DNA fragments are ligated into these vectors in easy 5-minute reaction on bench top
• Vector is designed for high-level expression of transient or stable GFP fusion proteins in wide range of mammalian cells
• Expression is easily detected in living cells using fluorescence
• Recombinant proteins expressed from these vector can be detected on western blots using GFP Antiserum

Cell Analysis, Cell-Based Reporter Assays, Cellular Imaging, Constitutive Expression, Enzyme and Protein Activity Assays, Fluorescent Protein (e.g. GFP) Assays, Fluorescent Protein Assays (GFP)/Mammalian Expression, Protein Expression, Proteins, Expression, Isolation and Analysis, Reporter Assays

Specifications

• Constitutive or Inducible System: Constitutive
• Delivery Type: Transfection
• Promoter: CMV
• Product Type: TOPO Expression Kit
• Reporter Gene: GFP (Cycle 3)
• Selection Agent (Eukaryotic): Geneticin™ (G-418)
• Content And Storage: GFP Fusion TOPO™ Expression Kits contain two boxes. The GFP Fusion TOPO™ TA Cloning box contains linearized and topoisomerase I-activated cloning vector, dNTPs, salt solution control PCR template and primers, forward and reverse primers for sequencing and PCR screening, and a GFP expression control plasmid. Store at -20°C. The One Shot ¤ box contains transformation reagents including single-use, 50-μl aliquots of One Shot™ TOP10 Chemically Competent E. coli, S.O.C. medium, and supercoiled control plasmid. Store the One Shot™ box at -80°C. Kits are guaranteed stable for 6 months when properly stored.
• Protein Tag: GFP (Cycle 3)
• Cloning Method: TOPO-TA
• Quantity: 20 reactions
• Vector: TOPO-TA Vectors
• Product Line: TOPO
• For Use With (Application): Reporter Assays

Frequently Asked Questions (FAQs)

Can I use pBlue- or pGlow-TOPO reporter vectors to evaluate promoter function in E. coli?

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.

Do you offer a vector that I could use to do some promoter analysis studies?

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.

Can GFP fluorescence be detected in cells that have been stained for beta-galactosidase?

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.

What filter set should be used to detect Cycle 3 GFP fluorescence? How can I measure Cycle 3 GFP fluorescence using a fluorometer and what model fluorometer should I use?

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.

What are the recommended filter sets for detection of EmGFP, YFP, CFP, and BFP by fluorescence microscopy?

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.

What are the excitation and emission maxima for your fluorescent proteins (EmGFP, YFP, BFP, CFP, and Cycle 3 GFP)?

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

Are the fluorescent proteins you offer (EmGFP, YFP, CFP, BFP, and Cycle 3 GFP) humanized?

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

How does Cycle 3 GFP compare with EmGFP and EGFP?

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

How do your Vivid Colors fluorescent protein vectors compare to the previously sold Clontech BD Living Colors fluorescent protein vectors in terms of overall brightness?

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. 

I performed stable selection but my antibiotic-resistant clones do not express my gene of interest. What could have gone wrong?

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.

I used a mammalian expression vector but do not get any expression of my protein. Can you help me troubleshoot?

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.

I am using a mammalian expression vector that has the neomycin resistance gene. Can I use neomycin for stable selection in mammalian cells?

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.

Is it okay if my construct has an ATG that is upstream of the ATG in my gene of interest? Will it interfere with translation of my gene?

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.

Do you offer a GFP-expressing mammalian expression vector that I can use as a control to monitor my transfection and expression?

We offer pJTI R4 Exp CMV EmGFP pA Vector, Cat. No. A14146, which you can use to monitor your transfection and expression.

I am working with a mouse cell line and would like to express my gene at high levels using one of your vectors with the CMV promoter. Do you foresee any problems with this approach?

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.

Do I need to include a consensus Kozak sequence when I clone my gene of interest into one of your mammalian expression vectors?

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.

Do I need to include a ribosomal binding site (RBS/Shine Dalgarno sequence) or Kozak sequence when I clone my gene of interest?

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.

Can you tell me the difference between a Shine-Dalgarno sequence and a Kozak sequence?

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.

What is the consensus Kozak sequence and what is the function of the Kozak sequence?

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.

For Research Use Only. Not for use in diagnostic procedures.