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IPTG Protein Induction & Extraction Protocol2
IPTG Protein Induction & Extraction Protocol2
Modified from IPTG Induction and Extraction of Proteins from Bacteria, by Swathi
Arur and Sudhir Nayak, Schedl Lab. Washington University, St. Louis.
Introduction
Induction in bacteria can be performed using one of two basic methods. Fast induction will not work for
all proteins and may give you suboptimal yields. Slow induction may enhance the solubility of some
proteins. The best method for your research will depend on your particular protein and the application.
If you want optimal solubility both should be tested before scaling up. This protocol is generalized and
optimal conditions may vary based on factors such as the bacterial strain, recombinant protein, and
parent plasmid.
Method
Fast induction
1. From a relatively fresh plate, pick a colony and grow overnight at 30C (or 37C) in 1-2 ml
LB+Antibiotic (e.g. Ampicillin, Kanamycin, Carbenicillen, etc.) in a 15 ml tube on a rotator or
shaker.
2. Dilute 1:50 (1:100 if 37C overnight) in 2 ml LB+Antibiotic and grow 3-4 hours at 37C in 15 ml
tube in a rotator.
3. Prepare 1 ml LB+Antibiotic+1mM IPTG (GoldBio Catalog # I2481) in a 15 ml conical and prewarm
to 37C about 10 minutes before use.
4. After 3-4 hours remove 1 ml from tubes at 37C and place in labeled 1.5 ml tubes. Centrifuge at
maximum speed for 30 seconds at RT and remove supernatant. Freeze pellet at -20C until
needed. THIS IS THE UNINDUCED CONTROL.
5. Add 1 ml prewarmed (37C) LB+Antibiotic+1mM IPTG to 15ml tube and return to 37C for 3-4
hours. This will get the final volume back to 2 ml and the final concentration of IPTG to 0.5mM.
6. After 3-4 hours, transfer 1 ml from the induced sample to labeled 1.5 ml tubes and centrifuge at
maximum speed for 30 seconds at RT and remove supernatant. Freeze pellet at -20C until
needed. THIS IS THE INDUCED SAMPLE.
7. Sample preparation for SDS-PAGE: Add 100 l of 1X Loading Buffer (see Solutions) with 1% BME
to uninduced and induced samples. Vortex the samples for 10 seconds to 1 minute or until
there are no clumps of bacteria. Boil the samples for 3 to 5 minutes. Centrifuge at maximum
speed for 30 seconds at RT and load 5-25 l (usually 10 l) depending on gel (amount of protein,
size of pellet, Western, etc.).
Slow induction
For slow induction of protein follow fast induction protocol with the following changes:
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Gold Biotechnology
Web: www.goldbio.com
St. Louis, MO
Ph: (314) 890-8778
email: contactgoldbio86@goldbio.com
Solutions
Loading Buffer - 4X Stock (to make total volume 40 ml)
50mM Tris-HCl; pH 6.8 (Tris-HCl, GoldBio Catalog # T-095) (2.0 ml 1M Tris-HCl; pH 6.8)
2% SDS (0.8 g SDS)
10% Glycerol (4.0 ml 100% Glycerol)
12.5mM EDTA (EDTA Disodium, GoldBio Catalog # E-210) (1.0 ml 0.5M EDTA)
0.02 % Bromophenol Blue (Bromophenol Blue, GoldBio Catalog # B-092) (8 mg Bromophenol
Blue)
Fill to volume with dH2O (~33 ml dH2O)
Gold Biotechnology
Web: www.goldbio.com
St. Louis, MO
Ph: (314) 890-8778
email: contactgoldbio86@goldbio.com