Protein Induction with IPTG

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Shannon
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Protein Induction with IPTG

Hi.
I'm a student trying who is just starting out in lab and I have so much more to learn. I've got some doubts to clear. Hope you guys can help me.
I'm currently trying to purify a protein whose protein expression level is rather low. But the instructions I got from my sup isn't very clear and I would like try and optimize the condition for protein production without only having to increase the volume.
1. I read in one of the topic that IPTG induction is carried for no more than 5 hours. But what I was doing is to leave it overnight, usually for 18hrs. Is it too long? If I'm going to try vary the concentration of IPTG to find the optimal value, how long incubation time could you suggest?
2. Does the time of induction affect protein production? I was taught that if you are working with small volume, it is alright to directly add IPTG right from start. But for large scale, it is to be added when the OD reaches around 0.7. Could someone explain to me?
3. As my protein of interest has a His-tag, will the incubation time with nickel beads be a factor worth considering and how about the amount of nickel beads added?
I'm not sure if I've phrase my question sensibly, but I hope they make some sense.

Ivan Delgado
Ivan Delgado's picture
 

 
Hi Shannon,
Your questions are very much valid since the optimization of protein expression and purification can be a very tricky business. If you are lucky things work out using standard conditions, but invariably there are a number of proteins that simply do not like to be expressed. Here are some answers to your questions to get started: 
1. Incubation time will vary, with as little as 2 hours being enough for highly expressed proteins, while overnight may be necessary for proteins that simply do not express very well (but are stable enough to survive the long incubation). For more details about this and many other points look at my protocol page for this experiment. If you are so inclined, you may want to do a quick experiment where you test the expression of your protein at multiple times (2 hr, 4 hr, 8 hr, and overnight). Then choose the best one.
Together with the incubation time is the temperature at which you grow your cells. While 37oC will give you nice, fast, robust growth of your culture, sometimes growing your cells at 28oC is better, mainly because at a slower growth rate proteins that may not fold well during expression will have an easier time folding, thus increasing your yield (and minimizing inclusion bodies, which is what happens when you protein is expressed and precipitates out of solution)
2. Incubation time is important. The reason why people say that you should induce at an OD600 of 0.7 is that cells at that OD have reached exponential growth. In other words, the vast majority of cells are alive and healthy, which makes them ideal for protein expression. If you go to high (over 1.0 or higher) your culture will start collecting dead cells, which cannot express protein. A too low OD is also not good (<0.4) because most of your culture is media so there aren't enough cells to make enough protein
3. The interaction between a His-tag and a Nickel column is something that should occur pretty fast. In my experience 30 minutes is more than enough (see this protocol). Yet, again if you are so inclined you may want to perform a couple of experiments to determine if incubation time affects your yield. I would do something  like 30 minutes and 2 hours. If you see a significant increase in protein yield at 2 hours compared to 30 minutes, which is unlikely, you could test even longer hours. But most likely 30 minutes will be more than enough. 
Good luck
 

Shannon
Shannon's picture
Hi Ivan.

Hi Ivan.
Thanks for your clear explanation. I'm now going to design my experiment.
 

annivsahra_p
annivsahra_p's picture
Dear MR.Ivan,

Dear MR.Ivan,

Its nice and very good explanation. I have one doubt on induction with IPTG. After the cells has been induced with IPTG, we are monitoring the density of the cells growth. Before the induction , we may have some O.D like 0.7, after the induction the O.D will increase right, when the O.D ceased or decreased after few hours, then we can stop the experiment and go for further pellet collections. So my question is instead of going for overnight induction , we can monitor through the cells density.

Ivan Delgado
Ivan Delgado's picture
Hi annivsahra,

Hi annivsahra,

You can definitely monitor your cells after IPTG induction by measuring OD increase/decrease. The only reason why I suggested overnight growth after induction was because it is convenient. Anything you can do to maximize the ideal conditions of your protein induction, including even collecting aliquots of your cells at different times and extracting your protein to determine which is the best time for expression, would be advantageous. 

Good luck

annivsahra_p
annivsahra_p's picture
Dear Mr.Ivan

Dear Mr.Ivan

Its really brilliant and great idea. I have more doubts on protein purification .I would like to clear my doubts , if you feel free.

Ivan Delgado
Ivan Delgado's picture
 

 
Feel free to post any questions you have annivsahra. We will answer them as best we can. 

Cheers

annivsahra_p
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Dear Mr/ Dr.Ivan.

Dear Mr/ Dr.Ivan.

                                Thanks for your kind concern. I have one doubt on Cell lysis. We are using frech press for cell lysis. I would like to know , how to ensure the cell has been lysed or not , We are following microscopy to see the cell lysis, apart from this , any other techiniques are there to identify the how many precentage of cell has been lysed.

Ivan Delgado
Ivan Delgado's picture
A while back I performed a

A while back I performed a study where I tested different methods of lysing cells for protein isolation. Using a french press was by far the best method to obtain as complete lysis as you can, so you are using the best method I am aware of. 

As for making sure that you are getting full lysis, as long as you are using the french press the right way you should be getting full lysis. If you are concerned that some bacteria may be surviving a pass through the french press, which is possible if you are overloading the press with too much bacteria, then you could simply pass your cells through the press a second time (or not use too much bacteria, which is hard to do anyway, you would need to really grow a very dense culture). It is close to impossible for a bacteria to survive two passes through a french press, although normally one pass is more than enough.

annivsahra_p
annivsahra_p's picture
Dear Mr / Dr.Ivan.

Dear Mr / Dr.Ivan.

                        Thanks for your kind consideration . In frech press we will going for three cycles or pass. I would like to konw how to confrom that , the cell has been lysed . Because in first cycle the i will check the lysed sample in microscopy , where we can find some few cells in full form and second cycle as same and same in the third pass too .. i would like to ensure that . 1. How to confrom that how many percentage of cell has been lysed. and 2. is there any other technique to find the whether is cell is lysed or not . Please confrom the same .

manti kumar saha
manti kumar  saha's picture
Microbiology

Dear Mr./Dr. Annivsahra,if u are working with bacteria, then you can do spread plating with your control and test to analyse the efficiency of French press in lysing the cell...

manti kumar saha
manti kumar  saha's picture
Microbiology

Dear Mr./Dr. Annivsahra,if u are working with bacteria, then you can do spread plating with your control and test to analyse the efficiency of French press in lysing the cell...

Ivan Delgado
Ivan Delgado's picture
 

 
To be honest I never really went into that much detail as far as determining how many cells remained whole after french press. I think that microscopy is a perfectly fine way of checking. Beyond that I am not sure. 

annivsahra_p
annivsahra_p's picture
Dear Mr/Dr.Ivan.

Dear Mr/Dr.Ivan.

                            I am sorry for distrubing you , Thank you v ery much for your kind reply . But i have worked on it with cell lysis , i have done an experiment with cell lysis samples. I have centrifuged the cell lysis sample at 11000 rpm, i have done quantitative analysis with UV-Vis spectrophotometer, in which i have found that, the live cells won't show any absorbance at 260 or 280nm. i have checked the centrifuged samples, the ratio of 260/280 is seems to be 1.6 at first cycle and in second cycle it shows 1.2 and in third cycle it shows 0.92 . i have checked the all samples in SDS page its gave an clear cut idea. this gave me an idea to find the percentage of cell lysed during the cycle not in exact but apporximate.

                   I would like to know , whether the above mentioned statement is right or wrong.

Ivan Delgado
Ivan Delgado's picture
The types of analysis you

The types of analysis you performed to assess how well your cells were lysed is very complete. I think you can assume that most of your cells were lysed, which in the end is all you really need. So in that light you can say your statement is right.

dccup43
dccup43's picture
Hi Shanon,

Hi Shanon,

You can also check OD at 600nm,to ensure whether your cells are lysed(Bacterial cells) or not after every passage.

annivsahra_p
annivsahra_p's picture
Dear Mr./Dr. Dccup,

Dear Mr./Dr. Dccup,

                            I strongly agree with your words. Before that i would like to confrom that, live bacterial cell and lysed bacterial cells can able to show absorbance at 600 nm.

srinivas2201
srinivas2201's picture
After doing cell lysis on

After doing cell lysis on french press ,, one time you cna do sonication too .......

it is good to assure that all cell has been completly lysised !

Andreas Heinemann
Andreas Heinemann's picture
Hello,

Hello,

if you need any help, support or parts for FrenchPress, feel free to contact me directly - may be you have followed the other discussion regarding the good old French Press
Just visit   www.frenchpress.de

Good luck for your sciences

ratno
ratno's picture
 After incubate with IPTG for

 After incubate with IPTG for 2-3 hours, could we keep the E. coli in 4 C for overnight and to continue next step on the next day?

Chin Fen Teo
Chin Fen Teo's picture
Hi ratno,

Hi ratno,

Threorically, yes-- but at your own risk of having protein degradation. (hence I won't recommend anyone to do that)

That being said, the best way is to spin down the bugs- Once you have the pellet, you can store the pellet in the freezer (-20degC or -80degC) until the next step.

Good luck.

Pradeep Tekam
Pradeep Tekam's picture
BIOTECH

hi everyone I want to know that whether it is neccessory to induce the cell with IPTG while i am optimizing the OD vs WCM condition?Thank you.