hi, i have expressed a protein that is rich in cysteine residues, i hazve tried to purify it but it is expressed in the inclusion bodies. since rich in cysteine and having a small half life refolding is very difficult. my protein specifically binds to cadmiun ions. i have grown it at lower temperatures 18-25 degree celcius in order to get it into cytosolic fraction but no benefit.
anybody could tell me how to get it inside the cytosol. any suggesstions are welcomed
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#2
bhavana
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Posted 12 September 2010 - 04:06 AM
Alog with the temp u should also standardize the concentration of IPTG to be used
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bhavana
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Posted 12 September 2010 - 04:07 AM
Along with the temp u should also standardize the concentration of IPTG to be used
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sdar
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Posted 14 September 2010 - 10:11 PM
COULD YOU PLEASE TELL ME I SHOULD INCREASE OR DECREASE THE IPTG CONCENTRATION BECAUSE AT 37 DEGREE CELSIUS IT GIVES MAX EXPRESSION AT O.O5mM IPTG
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Adrian K
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Posted 15 September 2010 - 04:40 AM
something is unclear here.
so you mean that the MAX Expression is at 0.05mM IPTG at 37C but is in inclusion body?
If so, I suggest you to further reduce your IPTG to 0.025mM IPTG, and keep your cultures at room temp (i assume yours would be 25C) or at 30C.
IMHO, 18-25C would be too cold for the bacteria to grow and produce efficient protein.
Also, change to rosetta strains might help. Just have to try all the possibilities.
Just my 2 cents.
p/s: using CAPS in forum makes your sentence hard to read.
so you mean that the MAX Expression is at 0.05mM IPTG at 37C but is in inclusion body?
If so, I suggest you to further reduce your IPTG to 0.025mM IPTG, and keep your cultures at room temp (i assume yours would be 25C) or at 30C.
IMHO, 18-25C would be too cold for the bacteria to grow and produce efficient protein.
Also, change to rosetta strains might help. Just have to try all the possibilities.
Just my 2 cents.
p/s: using CAPS in forum makes your sentence hard to read.
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..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
