I am purifying AdhE (96 kDa) protein which is bifunctional and ontains 2 functional domains; (acetaldehyde dehydrogenase and Alcohol dehydrogenase). Before purification, I did a small scale protein expression test. Th econditions are as below:
All conditions were set after reaching an OD of 0.6nm.
500ul of the samples were collected prior and after induction of the proteins, spun down at max speed for 5 mins and the supernatants were duscarded. The cell pellets were then suspended in 40 ul of Buffer (20 mM imidazole, 400 mM NaCl and 0.05% tween 20), 25ul of loading SDS buffer and heated at 95C for 10 mins. 5ul of samples were loaded on to the gel.
My results were such that, the bands of my protein of interest was not visible after induction. Only for AIM samples, there was a thick smear of protein bands after induction however, no one singular protein band resembling my protein.
Any ideas why my expression have not worked?
The picture shows post induction in AIM Media
Honestly, I can see only smear at wells 3,4,5 (induced samples) which is due to over loading. I give a trick, please measure the OD of the cultures, lets say the culture OD is 2 after overnight induction, take 250/2=125ul. do the same for all cultures. prior to induction it is 250/0.6=416ul. By doing this you are keeping the bacteria roughly constant. Lyse these mini cultures, with just 50ul whatever lysis buffer, spin down and load both soluble and insoluble (pellet) fractions. you would see a clear difference at least in pellet fraction, if at all there is induction of you GOI.
I agree that you should adjust the volume of cell culture which is loaded on the gel. Also, I can see a major band in the well 5 - this band is absent in other wells and can represent your protein of interest. Unfortunately, I am not sure at all, because the protein marker bands are not labeled, as well as the wells. Please, label them - in this case my answer would be more certain.
Also, could you, please, share your gels from other expression conditions?
Hi Liyana,
IPTG concentration plays a major role in over expression of proteins. I will suggest you to work with different conc. of IPTG( like from 0.1mM to 1mM) . temperature also is aan important constraint. for 18-25 degree celsius keep the induction time as 12 hours. if you are inducing at room temperature then atleast give 4-5 hours time.
Again proper cell lysis is also important, it may be possible that your protein might b expressing but since cell lysis is not done properly it cant be seen in SDS-PAGE. Try sonicating your sample in lysis buffer ( in ice for 15-20min) and then add loading buffer( for gel). You can also try loading both pellet and supernatant after cell lysis, as to see where your protein goes.
good luck!
this gel shows my expression pre and post induction using LB media and induction using IPTG.
This gel shows my expression post induction with AIM media. I put 5ul of each sample into the wells.
You have a lot of stuff sticking to the wells. It could be that your protein forms inclusion bodies that are not solubilized in SDS, and this is what you see at the top of the stacking gel. You could try adding 4 M urea to you samples to test whether you have such inclusion bodies.
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