What happens when we don't wash the Ni-NTA column? - (Jan/21/2008 )
I am tryin to purify a His-Taq protein.
It's my first time working with proteins so i have no idea what i am doing
I was given a Ni-Nta column and was told: PURIFY!
The first trial was fine
then i wanted to purify more but i didnt know we needed to wash the column
so i had just loaded the enzyme solution but it's taking so much more time
instead of 20 mins it's taking 3h for the wash buffer or what not to pass through the column
what happens if the column wasnt washed the previous time?
Don't i get a purified enzyme? should i redo it tomorrow after washing with NaOH?
thank in advance guys
-deedzzz-
QUOTE (deedzzz @ Jan 21 2008, 08:43 AM)
I am tryin to purify a His-Taq protein.
It's my first time working with proteins so i have no idea what i am doing
I was given a Ni-Nta column and was told: PURIFY!
The first trial was fine
then i wanted to purify more but i didnt know we needed to wash the column
so i had just loaded the enzyme solution but it's taking so much more time
instead of 20 mins it's taking 3h for the wash buffer or what not to pass through the column
what happens if the column wasnt washed the previous time?
Don't i get a purified enzyme? should i redo it tomorrow after washing with NaOH?
thank in advance guys
It's my first time working with proteins so i have no idea what i am doing
I was given a Ni-Nta column and was told: PURIFY!
The first trial was fine
then i wanted to purify more but i didnt know we needed to wash the column
so i had just loaded the enzyme solution but it's taking so much more time
instead of 20 mins it's taking 3h for the wash buffer or what not to pass through the column
what happens if the column wasnt washed the previous time?
Don't i get a purified enzyme? should i redo it tomorrow after washing with NaOH?
thank in advance guys
of a crude protein mix you will not purify a protein in a one-step column protocol f.i Ni-NTA; what you get is at least a good enrichment...
washing is necessary to lose unspecifically bound proteins; without washing, recovery is increased but enrichment decreased
-The Bearer-
the column also clogged and/or compressed. you need to clean and recharge the column.
-mdfenko-