O-064793 gibco tet test.indd

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Tetracycline-Tested GIBCO® FBS
The FBS Factor. Most cel culture systems stil employ
supplementation with FBS to promote cel growth. Any Increase control in tetracycline-induced expression given batch of FBS can contain serum from several thousand animals. The presence of tetracycline in a batch of serum is possible when administered to cattle prior to slaughter. As Inducible systems allow you to control the expression described earlier, low levels of tetracycline can cause expres- level of your gene of interest. Many of the systems cur- sion at an undesirable time. While we recommend the addi- rently available for regulating expression in mammalian tion of 1 µg/ml tetracycline to induce expression in a T-REx™ cells rely on tetracycline as the inducing agent, including cel line, the range of 0.1 µg/ml to 1 µg/ml can potential y Invitrogen’s T-REx™ System. These systems tend to be very sensitive, and expression may be activated in the presence The Tet-Tested GIBCO® FBS Solution. To address the
of a minimal amount of tetracycline. Therefore, it is impor- FBS factor, Invitrogen scientists have developed and vali- tant that the media and all media components, including dated an assay using reverse-phase HPLC with fluorescence fetal bovine serum (FBS), be free of tetracycline.
for detecting the presence of tetracycline in FBS. Using this How Regulation Works. The T-REx™ System uses
assay, as little as 19.7 ng/ml of tetracycline can be detected. regulatory elements from the E. coli tetracycline-resistance The assay was validated against three criteria: linearity, operon to manipulate expression from the CMV promoter. reproducibility, and sample stability (Table 1).
In the absence of tetracycline, the tetracycline repressor (TR) protein binds the promoter and prevents expression. tested for tetracycline (Figure 1). When using Invitrogen’s The addition of tetracycline causes the TR protein to release T-REx™ System and ordering FBS, you may request a lot from the promoter and al ows expression to proceed (1).
that has been tested for the presence of tetracycline. At the To achieve tightly controlled regulated expression, it is time of your order, our technical support team will review important that the uninduced system be tetracycline-free. the available inventory of the ordered FBS product and The lack of tetracycline ensures that expression of the gene select a lot tested for tetracycline that falls below the limit of interest is due only to the addition of defined quantities of detection. There is no additional charge for this service.
of tetracycline to the media. The ideal experiment would have little or no basal expression of the gene of interest. Consider the example in which a gene expresses a protein that produces undesirable effects within the cell (i.e. toxic- ity). The unknown presence of tetracycline in the culture system could potentially cause unwanted expression, lead- ing to misinterpretation of experimental results.
Table 1. The three criteria of the GIBCO® FBS tetracycline validation assay.
Description
The linear range for the assay is from 50 ng/ml to 1000 ng/ml. A correlation coefficient Reproducibility and accuracy at low levels are demonstrated by preparing the low standard 6 times. The %RSD of these samples needs to be 3.93% and 97.8% of the theoretical value.
Spiked samples do not show significant degradation after being freeze-thawed 3 times. Samples were stored at ambient temperatures unprotected from light for 4 hours.
QUEST | 29
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To order tetracycline-tested GIBCO® FBS, please visit worldwide (see page 103). For more information about the Figure 1. Sample HPLC data from a GIBCO® FBS tetra-
cycline assay.
T-REx™ System, please vProduct
Quantity Catalog No.
A. Tetracycline standard in FBS.
B. Tetracycline screen of a tetra-cycline-tested GIBCO® FBS lot.
T-REx™ Complete Kit with pcDNA™4/TO 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 T-REx™ Complete Kit with pcDNA™4/TO/myc-His etrac pycDNA™4/TO/myc-His
cline standard in FBS.
B. Tetracycline screen of a tetra-cycline-tested GIBCO® FBS lot.
Reference5.00
4.00 (1998) Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates induc Tetracycline - 13.414 ible gene expression in 3.00 cells. Human Gene Therapy 9(13): 1939-1950.
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 GIBCO® Certified and Qualified FBS products are routinely
assayed for the presence of tetracycline using reverse-phase
HPLC. A typical screen involves an FBS standard spiked with
tetracycline (A) and a sample from a lot of GIBCO® FBS (B). Note
the absence of the tetracycline spike in the lot being tested.
30 | QUEST

Source: http://tools.lifetechnologies.com/downloads/O064793_GIBCOTETTest.pdf