Troubleshooting: Protein Expression
Last time on Troubleshooting, we covered Tagged Protein Purification, but what happens if your prote...
IPTG is an analog of galactose that is non-metabolizable and inactivates the lac repressor to induce synthesis of β-galactosidase in E. coli. The expression of cloned genes under the control of the lac operon is induced by IPTG. It is also a substrate for thigalactoside transacetylase and has been reported to induce penicillinase in bacteria.
IPTG is commonly used in cloning procedures that require induction of β-galactosidase and is most often used with X-Gal (Gold Bio #X4281C) or Bluo-Gal (Gold Bio #B-673) for blue/white colony screening or Magenta-Gal (Gold Bio #B-378) for red/white colony screening of bacterial colonies.
IPTG is also used in the induction of recombinant proteins. In those systems, a protein of interest is encoded downstream of the IPTG inducible promoter. In the presence of IPTG, the protein of interest is induced in the cell culture. The culture can then be lysed and the protein expressed and purified through a number of methods, including His Tag or GST purification systems (for proteins with ligand tags).
GoldBio IPTG is made from a synthetic, non-animal origin.
Store powder IPTG desiccated at -20°C. Protect from light.
For IPTG
stock solution, store in 1 mL aliquots at -20°C for up to 1 year.
IPTG is readily dissolved in sterile water.
IPTG, which is similar to the lactose metabolite allolactose, induces the lac operon by binding to the lac repressor. Once IPTG binds to the lac repressor, a conformational change occurs, causing the lac repressor to dissociate from the lac promoter. Once the lac repressor dissociates, RNA polymerase binds to the promoter to initiate expression.
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Most papers recommend using between 0.1-1.0mM IPTG for expression. However, the amount you should use depends on your proteins. However, it is best to run an IPTG titration with ranges between 0.01-1.0mM and compare using SDS-PAGE to determine the optimal concentration. Something to keep in mind is higher concentrations of IPTG can cause inclusion bodies.
IPTG is non-metabolizable, so its concentration remains stable and doesn’t get consumed by the bacteria.
You should usually add IPTG at the mid-log phase (OD600 ≈ 0.4–0.6) to ensure cells are healthy and dividing rapidly.
No—filter sterilize only. IPTG is heat-sensitive and should not be autoclaved.
IPTG is stable when stored properly (aliquoted at -20°C, protected from light), but repeated freeze-thaw cycles should be avoided.
The best practice is to use single-use aliquots of IPTG. Do not refreeze IPTG leftovers to avoid degradation or contamination.
IPTG can be toxic at high concentrations or in sensitive strains. Lowering the IPTG concentration or using auto-induction media can help.
Catalog ID | I2481 |
---|---|
Name(s) |
IPTG Isopropyl-beta-D-thiogalactoside |
CAS # | 367-93-1 |
Formula | C9H18O5S |
MW | 238.30 g/mol |
Grade | MOLECULAR BIOLOGY GRADE |
Storage/Handling | Store desiccated at -20°C. Protect from light. |
PubChem Chemical ID | 656894 |
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