Tetracycline Repressor (TetR)

Tetracycline is an antibiotic which has the ability to inhibit the growth of wide variety of organisms, including gram-positive and gram-negative bacteria, rickettsias, mycoplasmas, chlamydias, and certain viruses, protozoa, and actinomycetes. It interferes with the production of proteins that the bacteria need to multiply and divide (bacteriostatic). Tetracycline Hydrochloride mode of action is as a protein synthesis inhibitor via an aminoacyl-tRNA binding mechanism to the 30S subunit. Mode of resistance is the loss of cell wall permeability.\n\nThe 23kD Tet repressor protein (TetR) regulates transcription of a family of tetracycline resistance determinants in Gram-negative bacteria. The Tet system is the most widely used regulatory system for conditional gene expression in eukaryotic cells. TetR can be used for that purpose in some organisms without further modifications. In mammals and in a large variety of other organisms, however, eukaryotic transcriptional activator or repressor domains are fused to TetR to turn it into an efficient regulator.\n\nThe excessive use of antibiotics has enabled bacteria to develop resistance through a variety of mechanisms. The most common bacteriostatic action of the broad-spectrum antibiotic tetracycline (Tc) is by the inactivation of the bacterial ribosome so that the protein biosynthesis is interrupted and the bacteria die. The most common mechanism of resistance in gram-negative bacteria against Tc is associated with the membrane-intrinsic protein TetA, which exports invaded Tc out of the bacterial cell before it can attack its target, the ribosome. The expression of TetA is tightly regulated by the homodimeric Tet repressor (TetR)(2), which binds specifically with two helix-turn-helix motifs of operator DNA (tetO; K(ass) approximately 10(11) M(-1)) located upstream from the tetA gene on a plasmid or transposon. When Tc diffuses into the cell it chelates Mg(2+) and the complex [MgTc](+) binds to (TetR)(2) to form the induced complex (TetR small middle dot[MgTc](+))(2). This process is associated with conformational changes, which sharply reduce the affinity of (TetR)(2) to tetO, so that expression of TetA can take place, thus conferring resistance to the bacteria cells against Tc. Crystallographic studies show sequence-specific protein-nucleic acid interactions in the (TetR)(2) small middle dottetO complex and how the binding of two [MgTc](+) to (TetR)(2) enforces conformational changes that are stabilized by cooperative binding of two chains of eight water molecules each so that the formed (TetR small middle dot[MgTc](+))(2) is no longer able to recognize and bind to tetO. Since the switching mechanisms of the TetR/[MgTc](+) system is so tight, it has proven very useful in the regulation of eukaryotic gene expression and may also be applicable in gene therapy.\n\nApplications: \nSuitable for use in ELISA, Western Blotting. Other applications not tested.\n\nRecommended Dilution:\nELISA: 0.2ng detection limit\nWestern Blotting: 1:1000. 0.8ng detection limit. Recognizes a 24kD iprotein.\nOptimal dilutions to be determined by the researcher.\n\nStorage and Stability:\nMay be stored at 4 degrees C for short-term only. For long-term storage and to avoid repeated freezing and thawing, aliquot and add glycerol (40-50%. Freeze at -20 degrees C or colder. Aliquots are stable for at least 12 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

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SPECIFICATIONS

Catalog Number

T2965-35B

Size

100ug

Applications

ELISA, WB

Hosts

Rabbit

Reactivities

Yeast/Fun

Form

Supplied as a liquid in PBS, 0.02% sodium azide.

P Type

Pab

Purity

Purified

References

General References:\n1. Pook, E., et al., Eur. J. Biochem. 258:915-922 (1998)\n2. Hillen, W. and Berens, C., Annu. Rev. Microbiol. 48:345-369 (1994)\n3. Gatz, C., et al., Biospektrum 1:23-29 (1995)\n4. Mayford, M., et al., Science 274:1678 (1996)\n5. Angew Chem Int Ed Engl. 2000 Jun 16;39(12):2042-2052

Additional Info

Recognizes the TetR protein. Used to monitor the regulation of gene expression in mammalian cells. The TetR antibody is not reactive to human or other mammalian proteins.