Frequently Asked Questions
1. What if my protein does not express?
- Try using a higher concentration of The Inducer™.
- Run verses normal conditions to see if plasmid is still in the bacteria.
- Expression plasmid used must contain a T7 lac promoter.
2. What if my protein is still insoluble?
- Try using less of The Inducer, 1.0-2.0 grams/liter.
- Reduce the incubation temperature.
- Add more DTT to the lysis buffer or another reducing agent.
3. My protein expresses but the yield is low?
- This is expected, The Inducer™ will not yield as much total protein or protein of interest but more should appear in the soluble fraction.
Background:
Recent functional genomic studies have been used to identify thousands of novel cDNAs and their corresponding proteins. As many researchers are aware, expressing these proteins in E. coli using IPTG can be filled with difficulties. One of the most persistent and challenging problems is the expression of protein that forms insoluble inclusion bodies that cannot be easily purified. Extracting insoluble protein under non-denaturing conditions using detergents and sonication can be moderately successful, but is time consuming and yields low levels of protein. Extracting the protein under denaturing conditions with urea or other reagents is often successful, but using these methods requires refolding the protein that may result in a loss of structure and activity.
Perhaps the easiest method for generating appreciable amounts of soluble protein in E. coli is to create conditions that reduce the rate of expression. Theoretically, lowering the rate allows the protein to fold properly and remain in solution so that inclusion bodies never form. Achieving a slower rate of expression has been attempted by reducing incubation temperatures and IPTG concentrations, but these manipulations, as most researchers realize, yield marginal increases in the amount of soluble protein.
Here we present a product called The Inducer that was specifically designed to lower the rate of expression compared with IPTG in E. coli protein expression systems. Data indicate that induction with The Inducer™ has been shown to yield increased levels of soluble protein compared with expression using IPTG.
Catalog # Price/Unit
