I have solubilized my protein with 0.3% sarcosine and purified by using Ni-NTA,during purification most protein is going into flow through.
I have diluted my sonicated sample to 0.1% sarcosine but still I am unable to get binding of protein.
Hi Santosh!!
My suggestion for you is that you can do 2 things. First try to optimize the percentage of sarcosine which makes your protein soluble (soluble might be 50% but try to take less percentage of sarcosine) then the second thing take mass culture of around 1 liter and sonicate it with optimize percentage of sarcosine (add sracosine only in your lysis buffer avoid sarcosine in wash and elution buffer). sonicate your sample (here you have to optimize the time for sonication as well with proper pulse on & off). After sonication centrifuge the sample in maximum rpm for 30 mins in 4 degree, then keep your sample for dialysis in dialysis tube for overnight.change the buffer (you can use the same buffer composition without sarcosine) 2-3 times.next day you purify your protein.i Did this many times and it works. Take small column for purification.we use qiagen plasmid midi column (used one) to purify the protein from NI-NTA matrix..
You should not give more wash with imidazole..just give wash with10 ml each of 20mM imidazole and 40mM imidazole wash buffer then elute your protein in 200mM imidazole.
And very first confirm your recombinant protein with western blot or purify in denaturing condition using urea, it makes you confirm about your recombinant protein.
Is this a membrane protein? If so you need detergent throughout the purification the CMC of sarcosine is ~14mM (0.4%), also why sarcosine?
Thanks for ur suggestions Mr.Gaurav...
It is not a membrane protein....
I also agree that sarcosine should be removed before IMAC purification, but also using Ni-IDA instead of Ni-NTA may help - Ni-IDA provides more biding sites for protein binding.
Thank u Marcela...
@Gaurav
After dialysis yield of my protein was increased in elution...
Thanks for sharing your experience..
Gaurav Chhetri Sir, I used this approach as well my protein is soluble in 1% sarcosine , but when i perform dialysis my protein get precipitated resulting in total loss of protein, what should I do
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