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.
Product Specifications:
IPTG
Isopropyl-beta-D-thiogalactoside
MOLECULAR BIOLOGY GRADE
Forumla: C9H18O5S
MW: 238.30 g/mol
Purity: >99% by HPLC
Dioxane free
Storage/Handling: Store desiccated at -20°C. Protect from light.
PubChem Chemical ID: 656894
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.
Click to expand image and see full detail.
Most papers recommend using between 0.1mM – 1.0 mM 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 0.01mM – 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 can be dissolved in sterile water.
To make fresh 100mM IPTG stock solution:
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.
Primary applications of IPTG include:
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. |
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