pTrcHis TOPO™ TA Expression Kit
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Citações e referências (1)
Invitrogen™

pTrcHis TOPO™ TA Expression Kit

The pTrcHis- and pTrcHis2-TOPO™ TA Expression Kits allow fast, efficient cloning of PCR products into a prokaryotic expression vector forLeia mais
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Número do catálogoQuantity
K44100120 reactions
Número do catálogo K441001
Preço (BRL)
-
Quantity:
20 reactions
The pTrcHis- and pTrcHis2-TOPO™ TA Expression Kits allow fast, efficient cloning of PCR products into a prokaryotic expression vector for high-level, regulated expression from the trc promoter. The vector in each kit, pTrcHis-TOPO™ or pTrcHis2-TOPO™, includes a minicistron element for enhanced translation efficiency of eukaryotic proteins in E. coli. Both vectors are provided linearized and activated with topoisomerase I for 5-minute TOPO™ Cloning with >85% cloning efficiency. To simplify protein purification and detection, the pTrcHis-TOPO™ and pTrcHis2-TOPO™ vectors include the following features:

pTrcHis-TOPO™

• N-terminal Xpress™ epitope for detection with an Anti-Xpress™ Antibody
• N-terminal polyhistidine (6xHis) tag for purification using nickelchelating resin and detection with an Anti-HisG Antibody
• Enterokinase cleavage site for efficient removal of N-terminal fusion tags

pTrcHis2-TOPO™
• C-terminal c-myc epitope for detection with an Anti-myc Antibody
• C-terminal polyhistidine (6xHis) tag for purification using nickel-chelating resin and detection with an Anti-His(C-term) Antibody
For Research Use Only. Not for use in diagnostic procedures.
Especificações
Antibiotic Resistance BacterialAmpicillin (AmpR)
Bacterial or Yeast StrainTOP10
CleavageEK (Enterokinase) Recognition Site
Constitutive or Inducible SystemInducible
Expression MechanismCell-Based Expression
Expression SystemE. coli
Inducing AgentIPTG
Product TypeTA Expression Kit
Quantity20 reactions
Selection Agent (Eukaryotic)None
VectorpTrc
Cloning MethodTOPO-TA
Product LineTOPO
PromoterTrc, lacO
Protein TagHis Tag (6x), Xpress Epitope Tag
Unit SizeEach
Conteúdo e armazenamento
The pTrcHis- and pTrcHis2-TOPO™ TA Expression Kits each contain two boxes. The TOPO™ TA box contains 200 ng of linearized, topoisomerase I-activated pTrcHis-TOPO™ or pTrcHis2-TOPO™ vector, sterile water, dNTPs, 10X PCR buffer, a control template and primers, primers for sequencing or PCR screening, and an expression control plasmid. The One Shot™ TOP10 box contains twenty-one, 50-μl aliquots of chemically competent TOP10 E. coli, S.O.C. medium, and a supercoiled control plasmid. Store the TOPO TA Cloning™ box at -20°C. Store the One Shot™ box at -80°C. All reagents are guaranteed stable for 6 months when properly stored.

Frequently asked questions (FAQs)

Can I store my competent E. coli in liquid nitrogen?

We do not recommend storing competent E. coli strains in liquid nitrogen as the extreme temperature can be harmful to the cells. Also, the plastic storage vials are not intended to withstand the extreme temperature and may crack or break.

How should I store my competent E. coli?

We recommend storing our competent E. coli strains at -80°C. Storage at warmer temperatures, even for a brief period of time, will significantly decrease transformation efficiency.

What are the advantages and/or disadvantages of using TOP10, DH5α, or other cloning strains versus BL21 Star (DE3) or BL21 (DE3) cells for expression with the pTrc system?

Top10, DH5α, other cloning strains

Advantages:
- Saves time, can go directly from cloning to expression.
- The glycerol stock is more stable because these strains are endA- and recA-.

Disdvantages:
- If GOI is toxic, the cloning step can be difficult.
- These cloning strains are not protease-deficient; therefore, the protein may be degraded.

BL21 Star(DE3) or BL21 (DE3)

Advantages:
- These expression strains are protease-deficient.
- You have to transform the plasmid into an expression strain.
- The glycerol stock may be unstable because these expression strains are not endA- and recA-.
- The (DE3) part is wasted because the promoter does not need T7 RNA polymerase.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What competent cell strain can I use for expression with my pTrc Expression System?

The pTrc promoter is recognized by E. coli RNA polymerase, not T7 polymerase, and therefore can be expressed in any E. coli strain, not just BL21 strains. Therefore, you can use Top10, DH5α, etc. for expression. However, if your gene is toxic, the cloning step can be difficult as there is leakiness in expression.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

How does glucose repress the pTrc promoter?

A transcriptional activator protein called CAP (catabolite activator protein) normally binds upstream of the trc promoter and activates transcription. This protein requires cAMP to bind the DNA. Adding glucose to the medium can reduce intracellular cAMP levels. Supplementing LB medium and agar plates with glucose will repress basal level transcription from the trc promoter. We recommend that you include 25 mM, 0.5% glucose in the selection medium to ensure stability of your insert.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

View all pTrcHis TOPO™ TA Expression Kit FAQs

Citações e referências (1)

Citações e referências
Abstract
The apoptotic regulatory protein ARC (apoptosis repressor with caspase recruitment domain) prevents oxidant stress-mediated cell death by preserving mitochondrial function.
Authors: Neuss M; Monticone R; Lundberg M S; Chesley A T; Fleck E; Crow M T;
Journal:J Biol Chem
PubMed ID:11438535
'ARC is an apoptotic regulatory protein expressed almost exclusively in myogenic cells. It contains a caspase recruitment domain (CARD) through which it has been shown to block the activation of some initiator caspases. Because ARC also blocks caspase-independent events associated with apoptosis, such as hypoxia-induced cytochrome c release, we examined ... More