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How to be good at prep? - Calling all protein experts (Dec/29/2010 )

I'll start by saying that I'm a noob/rookie tech.

I've been purifying 8 proteins of the same family using E Coli and standard vector with IPTG induction (pGEX, pMAL, etc), plus their point-mutated versions. Also, I have been purifying some other MBP tagged random protein for labmates.

Based on these proteins, honestly I could find no "general rule" for growing them, even for those that are closely related one grow at low temp, others grow at 37C, one must be induced with low IPTG, the other must be at high IPTG, some protein "behaves" well i.e. it isn't degraded fast, other proteins are difficult i.e. it degraded in the cell or in ice.

How are you going to get "good" at purifying protein, if there are so so many variables in preps? (good and efficient, e.g. by just predicting from its sequence you have general idea of what temp to grow, what induction, knowing what buffer to use, how fast, etc)

Or in reality there is no way to be "good" like I said above, and treat them as unique. Therefore everytime with new protein I have to:
1. read manual/papers that have purified them to determine strategy
2. from (1), do small scale growing/induction experiment to determine which is good, western blot
3. from (1 and 2), do small scale prep with diff buffers (e.g. use buffers that contain MgCl2 for protein that has been predicted to use it, vs no MgCl2)
4. rinse and repeat

Thanks

-MyProteinBulliedMe-

there is no general rule on how to succeed in protein expression and purification ...every protein is different!

with time you will gain more, more experience and you will increase your trouble-shooting capacities :) ...then things will go more smoothly

I sometimes use this programm for the prediction of protein solubility:
My link

this will give you a rough estimation on the solubility of your protein of interest (but do not take this for granted) ...if the prediction says very insoluble i would start with three different temps (37C, 25C and 16C), sometimes higher temps and a certain percentage of Ethanol is also good to increase solubility (due to induction of heat shock proteins).

Good luck and all the best!

Regards,
p

MyProteinBulliedMe on Wed Dec 29 08:47:26 2010 said:


I'll start by saying that I'm a noob/rookie tech.

I've been purifying 8 proteins of the same family using E Coli and standard vector with IPTG induction (pGEX, pMAL, etc), plus their point-mutated versions. Also, I have been purifying some other MBP tagged random protein for labmates.

Based on these proteins, honestly I could find no "general rule" for growing them, even for those that are closely related one grow at low temp, others grow at 37C, one must be induced with low IPTG, the other must be at high IPTG, some protein "behaves" well i.e. it isn't degraded fast, other proteins are difficult i.e. it degraded in the cell or in ice.

How are you going to get "good" at purifying protein, if there are so so many variables in preps? (good and efficient, e.g. by just predicting from its sequence you have general idea of what temp to grow, what induction, knowing what buffer to use, how fast, etc)

Or in reality there is no way to be "good" like I said above, and treat them as unique. Therefore everytime with new protein I have to:
1. read manual/papers that have purified them to determine strategy
2. from (1), do small scale growing/induction experiment to determine which is good, western blot
3. from (1 and 2), do small scale prep with diff buffers (e.g. use buffers that contain MgCl2 for protein that has been predicted to use it, vs no MgCl2)
4. rinse and repeat

Thanks

-pDNA-

Thanks pDNA, I'll try using that link. I guess since you also said it..then every protein is unique.

-MyProteinBulliedMe-