Both should be optimized. Usually it’s OD between 0.6-0.8, except if your protein is unstable, precipitates or has cleavage problems, in which case you can try a lower OD. IPTG depends on your protein and plasmid. For instance 0.2mM is recommended for pGEX, but I've seen many times that 0.05 is enough for a good quality sample for unstable proteins. For pQE 1mM is recommended, again I've seen that it works better with a lower concentration, 0.2mM in general. I always try 2 different colonies, 2 OD (for example 0.5 and 0.7), 3 IPTG concentrations (0.05, 0.2, 0.5), two temperatures (30 and 37°C), 2 times for induction (which depends on the stability of the protein, I’ve used between 30 min and 6 hours, lower when less stable). Each condition in 5ml tubes, comparing solubility and cleavage, after lysis and centrifugation by western blotting and SDS page.

0.8 OD is enough for induction. however you have to optimize your IPTG concentration and time required for induction ( try with 0.5mM to 4mM concentration of IPTG, 4 hours to overnight, check the expression @ every 2 hrs interval)

Both should be optimized. Usually it’s OD between 0.6-0.8, except if your protein is unstable, precipitates or has cleavage problems, in which case you can try a lower OD. IPTG depends on your protein and plasmid. For instance 0.2mM is recommended for pGEX, but I've seen many times that 0.05 is enough for a good quality sample for unstable proteins. For pQE 1mM is recommended, again I've seen that it works better with a lower concentration, 0.2mM in general. I always try 2 different colonies, 2 OD (for example 0.5 and 0.7), 3 IPTG concentrations (0.05, 0.2, 0.5), two temperatures (30 and 37°C), 2 times for induction (which depends on the stability of the protein, I’ve used between 30 min and 6 hours, lower when less stable). Each condition in 5ml tubes, comparing solubility and cleavage, after lysis and centrifugation by western blotting and SDS page.

Thank you all for the answers. Am using pGEX and I've tried using OD 0.6, 0.25mM IPTG and it worked. I've also reduced the temperature from 37oC to 30 oC and it also worked.

@Carola Bruna, could you share the results of your protein expression experiments?

During IPTG induction shacking is required or not? please let me know?

Yes it is required

OD600 should be in the range of 0.5 to 1.0 (exponential growth phase). I always give induction when my culture OD600 reaches beyond 0.5 and not more than 0.6. 

For the over-expression of recombinant proteins using IPTG induction, it is recommended to use IPTG in the range of 1 to 10 mM and the optimum concentration needs to be optimized. For me 0.05 and 0.1 mM IPTG works very well for E. coli BL21 (DE3) and I am getting good over-expression of the recombinant proteins on SDS-PAGE gel. 

Hello, 

Can anybody provide me the reference that why it is said that induce culture at 0.6 - 0.8 OD with 1 mM IPTG for overnight ??

Thank you

It highly depends on the expression vector and potein you are using. For pET28a(+) the optimal OD600 is 0.6. The optimal IPTG is dependend on the protein. I would make a induction test with 0.1 to 0.5 M IPTG. Just compare the expression level and the ammount soluble protein on a SDS-PAGE Scince IPTG is expancive and isn´t metabolised by the host I wouldn´t use more than 0.5 M.

Timm...I know you meant 0.5 mM.  I'd expect a nice curve with at least 3 conditions for both OD and IPTG concentrations, ultimately so you could near some model if necessary, albeit complicating stuff up.  Optimization is always the responsible way to go.

It depends of your protein but we have achieved very high volumetric productivities by inducing with IPTG for 4 hours near the stationary phase. Alternatively, you can opt to use auto-induction medium and just leave cells growing for 20-24 hours.

For IPTG induction: http://pubs.acs.org/doi/full/10.1021/bm5005564

and https://microbialcellfactories.biomedcentral.com/articles/10.1186/1475-2859-12-21

For auto-induction: https://amb-express.springeropen.com/articles/10.1186/2191-0855-3-11

Depends on the protein being expressed, plasmid and media used, process conditions and quality of the media components. Traditionally, induction is most commonly carried out with 0.5 - 1 mM IPTG during the exponential growth phase (approx. OD600nm~0.5-1) when cells are most actively dividing. However, our studies with the pET vector system indicates increased production with induction early in the stationary phase of growth (i.e OD600nm ~6-7) with little variation in production at IPTG concentrations between 0.1 - 1mM.  See the following publication for a comparison of process variables during batch production of proteins with the E.coli BL21(DE3)-pET expression system.

https://www.researchgate.net/publication/235749784_Batch_production_of_a_silk-elastin-like_protein_in_E_coli_BL21DE3_Key_parameters_for_optimisation

Article Batch production of a silk-elastin-like protein in E. coli B...

OD600=(0.6-0.8); [IPTG]=0.05M; 18 °C overnight on shaker.

It depends on medium you are growing your cells: In LB medium 0.6-0.8 is good density for indution. However, I have switched to TB medium, because it is more nutritious and is buffered it can sustain higher cell densities, so without too much worry I induce e.coli at 3-5 OD in TB. If using pET system I found that 0.1 mM IPTG and then growth ON at 18 C gives very good and robust expression even for tricky proteins.

P.S IPTG titration when using BL21 DE3 cells is a bit futile, because cells contain lac permease (lacY), which pumps IPTG/lactose against the concentration gradient. If you want to play with the IPTG, you should use Tuner DE3 cells, that lack this permease and thus intracellular lactose/IPTG concentration is proportional to concentration in medium

O.D. on 600 should be 0.6 and more but not than OD. 1, you can get 0.6 when shaking 250 rpm for 3.5 hours, ,this is for E.coli BL21(DE3)Pplys.

for induction i used 2 concentrations of IPTG, 0.1 and 0.4 mM for two temp. 20 C /over night, and 37C for 4 hours, all conditions gave gave over expression of my protein..you can use :

1. 250 rpm shaker

2. OD 600/0.6

3. IPTG 0.1/over night

4. 20 C

This is what you have to determine experimentally for your expression system. What is mentioned above are possible starting points for your own optimization.

Cells at OD600 = 0.6 are generally used for protein expression as the cells are at the log stage and you can expect high protein expression. The best way to go about expression study would be to optimise all the parameters like nutrient medium - you could go for LB, TB or 2XYT and check cells in which medium give best expression, incubation temperature after adding IPTG - you may go from say 16 degrees to 37 degrees, concentration of IPTG - you can select a range such as 0.25 mM to 1 mM, duration of incubation - you can keep the cells for 4 hrs if you are planning for a higher temperature like 30 or 37 degrees and overnight for 16 or 18 degrees.

You could also try out Auto induction media if you don't want your cells to grow under IPTG stress. You can vary the parameters accordingly for standardisation.

With 100 µg/mL Antibiotic, I shake 2 L flasks with 1 L of Terrific Broth media till OD600 ~ 0.9. Then I cool the flasks in a bucket of ice in 4°C cold room for one hour. I then do 200 µM IPTG induction, and shake the flasks at 16°C for 20 - 24 hours, 220 rpm until the optical density is about 7 - 10. I then lyse the cells with 6 passes through the homogenizer, and then isolate the supernatant by centrifuging the lysate at 17,000 rpm, 50 minutes, 4°C. (I probably should spin the cells longer.). The supernatant is really viscous and overloads a newly regenerated nickel column since I do His-tag protein purification usually. So the protein binds to the beads with repeated centrifugation to collect the beads with each addition of supernatant. About half of my soluble protein goes into the pellet, but 50% loss of protein from OD600 = 10 or about 10 trillion E.coli cells is a whole lot of recovery.

E.coli cells in LB shake flasks don't enter stationary phase until about 2 days and then they start to die and that is at 37 centigrade. In richer media and lower temperature that process is delayed.

Kram, Karin E, and Steven E Finkel. “Rich Medium Composition Affects Escherichia coli Survival, Glycation, and Mutation Frequency during Long-Term Batch Culture.” Applied and environmental microbiology vol. 81,13 (2015): 4442-50. doi:10.1128/AEM.00722-15

I believe that IPTG induction puts the cells under stress to produce lots of recombinant protein. So doing IPTG induction a little bit later (than convention) in the exponential phase allows attainment of higher cell densities since more cells have grown before induction.

0.2mM