Protein Soluble Test - Inclusion Bodies + UREA test? (Jan/18/2010 )
I'm doing my master's thesis and really don't know anything about anything... it's a good start Anyway, my ultimate goal is to create an antiserum against human protein. By far I have managed to clone the desired insert and the next step is purifying the protein... it's only a partial fragment and expressed in E.Coli, meaning by running SDS-PAGE my protein is in inclusion bodies.
Now... I'm supposed to make a protein soluble test with different urea concentrations. My supervisor just keeps saying how it's easy and I just throw different urea concentrations to the tubes... like great Is there any kind of protocol how it's actually really supposed to be performed? I have spent two days now searching some information... everybody is talking about it, but I really would need some detailed facts... like how long I'm supposed to centrifuge my samples at what speed and how much bacteria mass and what concentrations would be good to test?
I really would appreciate any help... as I feel really stupid. I'm not supposed to mind about kit protocols or anything as they have been made so that people can make money and I'm supposed to modify everything and yet, I don't have experience or even knowledge to do anything... I understand the basics... but still how? I think I'm never going to work as a biologist after this is over
Ok... I have been reading a lot Anyway, in theory am I able to see which urea concentration is going to make the protein soluble if I:
1. grow a small culture
2. take for example 1ml of culture
3. pellet cells (supernatant sample a)
4. resuspend the pellet with different urea concentrations
5. spin down
6. take supernatant (sample
7. add sds loading bugger
8. lyse by heating
9. run in gel
so if I repeat this with urea concentrations 1, 2, 4, 6, 8 M should I be able to see at which concentration the protein is soluble or am I really lost?
Juliasarmoire on Jan 18 2010, 10:14 PM said:
1. grow a small culture
2. take for example 1ml of culture
3. pellet cells (supernatant sample a)
4. resuspend the pellet with different urea concentrations
5. spin down
6. take supernatant (sample
7. add sds loading bugger
8. lyse by heating
9. run in gel
so if I repeat this with urea concentrations 1, 2, 4, 6, 8 M should I be able to see at which concentration the protein is soluble or am I really lost?
First of all, you probably only need to go up to 4M urea, because 6 and 8 will almost certainly totally denature the protein, if that's an issue for you. Usually 2-3M is enough to gently dissociate IBs, and the monomers remain folded.
Next, I would run both S/N and pellet side by side, so you can see the ratio of soluble to insoluble at each
Finally, I know SDS-PAGE can be frustrating, but do you really think it should be called "loading bugger"? . Sorry, couldn't help myself there.
swanny on Jan 18 2010, 05:30 PM said:
Juliasarmoire on Jan 18 2010, 10:14 PM said:
1. grow a small culture
2. take for example 1ml of culture
3. pellet cells (supernatant sample a)
4. resuspend the pellet with different urea concentrations
5. spin down
6. take supernatant (sample
7. add sds loading bugger
8. lyse by heating
9. run in gel
so if I repeat this with urea concentrations 1, 2, 4, 6, 8 M should I be able to see at which concentration the protein is soluble or am I really lost?
First of all, you probably only need to go up to 4M urea, because 6 and 8 will almost certainly totally denature the protein, if that's an issue for you. Usually 2-3M is enough to gently dissociate IBs, and the monomers remain folded.
Next, I would run both S/N and pellet side by side, so you can see the ratio of soluble to insoluble at each
Finally, I know SDS-PAGE can be frustrating, but do you really think it should be called "loading bugger"? . Sorry, couldn't help myself there.
Ahem, don't forget to lyse the sample before centrifugation! Depending on your Lab resources, you may be able to add commercially available lysis buffers.
B4 induction, take a sample of the cells for SDS-PAGE. Take a sample of the induced cells at harvest.
Lyse cells.
Spin down the lysate, decant/pipetman/ect. the soup into a new tube for SDS-PAGE.
The remaining pellet, which should be much smaller then the original cell pellet, is to be resuspended in 3M+ Urea and used for SDS-PAGE.
plz,
Try to do the math and keep the concentrations of each sample the same.