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Expressway™ Maxi Cell-Free E. coli Expression System with pEXP5-NT/TOPO™ and pEXP5-CT/TOPO™ vectors Invitrogen™

The Expressway™ Maxi Cell-Free E. coli Expression System uses an efficient, coupled transcription/translation reaction to produce milligram quantities of soluble, functionally active protein in 4-6 hours. The procedure can be performed in a single reaction tube and is easily scalable without the need for specialized equipment. The TOPO TA Cloning® expression vectors (5-min cloning with 95% efficiency) are provided for optimal expression results. In addition to all the advantages of an open expression system, Expressway™ Maxi technology provides a means to produce high levels of recombinant protein that may be easily detected and purified for various downstream applications.

Milligram level protein production within 4-6 hours
The key to the Expressway™ Maxi technology is the unique formulation of the Feed Buffer that boosts the reaction productivity so that more than one milligram of protein is produced in a 2-ml reaction within 4-6 hours (Figure 1). The formulation of the lysate enables protein synthesis using either circular or linear templates.

High-throughput (HTP) compatible
This system is also designed for HTP expression. One kit is good for 200 x 50-reactions, which can be accommodated by 2 x 96-well plates. Without feed buffer, such reactions need only two hour’s time. Once a positive expression is detected, scale-up protein production can be performed with the large Expressway™ cell-free format or in E. coli cell-based systems.

Optimized vectors
pEXP5 TOPO® TA vectors are optimized for use in the Expressway™ Milligram system (Figure 2). They are designed to minimize additional amino acid sequences that may interfere with native protein folding and functionality. Features of these vectors include:

• pEXP5-NT/TOPO® expression vector provides N-terminal histidine tag and TEV protease recognition site

• pEXP5-CT/TOPO® expression vector can be used for expressing proteins with a C-terminal histidine tag or for native protein expression by introducing a stop codon at the end of your gene of interest

Simple and fast procedure
The Expressway™ Maxi Cell-Free Expression System provides all the components needed for optimal cell-free protein production. The system includes an E. coli extract, IVPS reaction buffer, Feed Buffer, T7 enzyme mix, 19 amino acid mix, and individual tubes of methionine. To start a 2-ml reaction, mix the reaction buffer, 19 amino acid mix, your choice of labeled or unlabeled methionine and cysteine, T7 enzyme mix, and your DNA template (with T7 promoter) with the E. coli extract. After a 30-minute incubation, add the Feed Buffer. Milligram-levels of active protein will be synthesized within 4-6 hours. To perform HTP expression, simply premix all the component and aliquot into a HTP format.

TC-FlAsH™ TC-ReAsH™ II In-Cell Tetracysteine Tag Detection Kit, with Mammalian TC-Tag Gateway™ Expression Vectors (Green Fluorescence) (Red Fluorescence) Invitrogen™

TC-FlAsH™ TC-ReAsH™ II In-Cell Tetracysteine Tag Detection Kit is an expression tag-based fluorescence labeling system for live-cell labeling. The expression construct is created when the Gateway entry clone (containing the gene of interest) is recombined with one of two destination vectors (pcDNA6.2/nTC-Tag-DEST for N-terminal tagging or pcDNA6.2/cTC-Tag-DEST for C-terminal tagging). The expression construct is then used to transform the host strain. The protein can be detected either with green-fluorescent FlAsH-EDT2 reagent or red-fluorescent ReAsH-EDT2 reagent. The tag is very small (6 amino acids) and the protein of interest is fluorescent only when the labeling reagent is added. BAL wash buffer replaces the previously supplied Disperse Blue 3 and EDT-based wash buffer as an olfactorily more agreeable reagent that yields superior signal to noise.

The kit contains FlAsH-EDT2 labeling reagent, ReAsH-EDT2 labeling reagent, BAL wash buffer, pcDNA6.2/cTC-Tag-DEST, pcDNA6.2/nTC-Tag-DEST, and pcDNA6.2/nTC-Tag-p64 Control Plasmid. BAL wash buffer should be stored at 4°C, and the rest of the kit stored at -20°C, protected from light. The kit is stable for 6 months when stored correctly.

BenchPro™ 2100 Plasmid Purification Card and Reagent Tray Kit Invitrogen™

The BenchPro® 2100 Plasmid Purification Card and Reagent Tray Kit are used with the BenchPro® 2100 Plasmid Processing Station (Cat. No. MC1001) for large-scale purification of plasmid DNA from bacterial cells. The card is designed for a single-use, with a series of capture and purification membranes, to facilitate the purification of anion exchange–quality plasmid DNA starting directly from culture. The reagent tray is pre-packed and foil-sealed with all necessary buffers. Disposable cell liners and lids are also provided for adding bacterial culture into the instrument. No additional reagents or plasticware is needed for the plasmid purification.

pSCREEN-iT™/lacZ-DEST Gateway™ Vector Kit Invitrogen™

Using the pSCREEN-iT™/lacZ-DEST Gateway® Vector Kit, you can:

•Assess the potency of any RNAi knockdown reagent using a fast, sensitive, and cost-effective β-gal reporter assay
•Identify the best knockdown reagent for your RNAi experiments without knowing protein function, western blotting, or qRT-PCR

How it Works: The BLOCK-iT™ RNAi Target Screening system method uses a β-galactosidase activity assay to measure the knockdown ability of an RNAi reagent (Stealth RNAi™ siRNA, siRNA, dicer pools, or shRNA-containing plasmid). To identify the most effective knockdown reagent for your target, simply clone your target gene into the pSCREEN-iT™ /lacZ-DEST Gateway® vector. Co-transfect the resulting construct with the knockdown reagent being tested. Expressed genes will be fused to β-galactosidase, enabling you to use β-galactosidase levels to measure the amount of RNAi-induced degradation of the target gene (Figure 1). The more effective any specific knockdown reagent is at mediating RNAi, the more degradation of the fusion mRNA will occur, lowering β-gal activity (Figure 2).

The BLOCK-iT™ RNAi Target Screening System includes the pSCREEN-iT™/lacZ-DEST Gateway® vector for generating a construct containing a lacZ/gene of interest fusion. This vector features:

attR sites immediately following the lacZ ORF for fast and efficient recombination with any attL-flanked Gateway® entry vector to generate the lacZ/target gene fusions
•The CMV promoter for strong expression of the lacZ/target gene fusion, leading to increased assay sensitivity and better discrimination between knockdown reagents of varied effectiveness
•Positional cloning to fuse a gene or fragment to the 3´ end of the lacZ gene so that β-galactosidase will be expressed even if the target fragment being tested contains stop codons

In addition, the pSCREEN-iT™/lacZ-DEST vector is fully compatible with the Ultimate™ ORF Clone Collection. This makes a wide selection of potential target genes readily available for generating lacZ fusions.

Expressway™Lumio™ Expression and Detection System, without vector Invitrogen™

The Expressway™ Lumio™ Expression and Detection System takes advantage of the Lumio™ recognition sequence, a small, six amino acid sequence (Cys-Cys-Pro-Gly-Cys-Cys). The Lumio™ detection reagent binds the recognition sequence with high specificity and affinity, resulting in a bright fluorescent signal for real-time protein production analysis and immediate in-gel protein detection. In addition, Expressway’s specialized E. coli lysate, derived from a slyD mutant, eliminates nonspecific binding of the Lumio™ Green Detection Reagent to the endogenous SlyD protein and provides optimal background for detection of recombinant proteins (Figure 1). The Lumio™ Green Detection Kit is included in the system for real-time detection of protein synthesis as well as easy in-gel protein detection. The Lumio™ vectors (Figures 2 and 3) also feature:

attR sites for efficient recombination with any attL-flanked Gateway® entry vector
N-terminal Lumio™ tag (pEXP3-DEST vector) with TEV cleavage site for efficient removal of the Lumio™ sequence following purification
C-terminal Lumio™ tag (pEXP4-DEST vector) for easy, and immediate in-gel detection
T7 promoter, ribosome binding site, and T7 terminator optimally spaced for cell-free protein expression

Neutralization Solution for MagJET Plasmid DNA Kit Thermo Scientific™

Thermo Scientific MagJet Neutralization Solution is a component of the MagJET Plasmid DNA Kits (K2791/2792) and may be purchased separately. Provided in 25 mL aliquots.

Resuspension Solution for GeneJET Plasmid Maxiprep Kit Thermo Scientific™

Thermo Scientific GeneJET Resuspension Buffer is a component of the GeneJET Plasmid Maxiprep Kit (K0491/K0492) and may be purchased separately.

Elution Buffer for GeneJET Plasmid Midiprep Kit Thermo Scientific™

Thermo Scientific GeneJET Elution Buffer is a component of the GeneJET Plasmid Midiprep Kit and may be purchased separately. Provided in 60 mL aliquots.

Wash Buffer 1 for MagJET Plasmid DNA Kit (concentrated) Thermo Scientific™

Thermo Scientific MagJet Wash Buffer 1 (conc.) is a component of the MagJET Plasmid DNA Kits (K2791/2792) and may be purchased separately. Provided in 110 mL aliquots.

Wash Buffer 2 for MagJET Plasmid DNA Kit (concentrated) Thermo Scientific™

Thermo Scientific MagJet Wash Buffer 2 (conc.) is a component of the MagJET Plasmid DNA Kits (K2791/2792) and may be purchased separately. Provided in 60 mL aliquots.

Resuspension Solution for MagJET Plasmid DNA Kit Thermo Scientific™

Thermo Scientific MagJet Resuspension Solution is a component of the MagJET Plasmid DNA Kit (K2791) and may be purchased separately.

Lysis Buffer for MagJET Plasmid DNA Kit Thermo Scientific™

Thermo Scientific MagJET Lysis Buffer is a component of the MagJET Plasmid DNA Kits (K2791/2792) and may be purchased separately.
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