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protein purification - (Nov/29/2010 )

I have a basic question about the protein purification.....
I am expressing protein of 25 kDa in Bl21 strain with out any affinity tag, how can I decide that whcih column to use to purify the protein...
Either proceed to DEAE, Sp sephrose, Mono Q column???? give me some idea or reference material which can clear my mind about this.

best

-samita-

Hola, First you have to know how your protein is charged at the pH of work, Determine de isoelectric point (introduce the sequence in any pI calculator), and think that at any pH under the pI the protein is + charged and you need a ion exchanger charged - as Q or DEAE sepharoses, in the other hand if your protein has - charge you have to use S or CM sepharose. before prepare the colums of the main experiment, confirm the the fact in a eppendorf in batch with a low volume of resin 20-40 ul, wash, add loading solution, and check that your protein was bounded. If yes, make the big purification follow the instructions of the supplier. Good luck

-protolder-

protolder on Tue Nov 30 06:57:16 2010 said:


Hola, First you have to know how your protein is charged at the pH of work, Determine de isoelectric point (introduce the sequence in any pI calculator), and think that at any pH under the pI the protein is + charged and you need a ion exchanger charged - as Q or DEAE sepharoses, in the other hand if your protein has - charge you have to use S or CM sepharose. before prepare the colums of the main experiment, confirm the the fact in a eppendorf in batch with a low volume of resin 20-40 ul, wash, add loading solution, and check that your protein was bounded. If yes, make the big purification follow the instructions of the supplier. Good luck

you have the right idea but the exchangers backwards. use deae or q if the protein is negatively charged (pH above pI) and s or cm if the protein is positively charged (pH below pI).

-mdfenko-

samita on Mon Nov 29 22:53:28 2010 said:


I have a basic question about the protein purification.....
I am expressing protein of 25 kDa in Bl21 strain with out any affinity tag, how can I decide that whcih column to use to purify the protein...
Either proceed to DEAE, Sp sephrose, Mono Q column???? give me some idea or reference material which can clear my mind about this.

best


I would like to choose the easy way that was performed in previous studies. Your protein would be similar to some protein that is well known. Please search this way and find several papers of purification.

-newborn-

mdfenko on Tue Nov 30 20:02:36 2010 said:


protolder on Tue Nov 30 06:57:16 2010 said:


Hola, First you have to know how your protein is charged at the pH of work, Determine de isoelectric point (introduce the sequence in any pI calculator), and think that at any pH under the pI the protein is + charged and you need a ion exchanger charged - as Q or DEAE sepharoses, in the other hand if your protein has - charge you have to use S or CM sepharose. before prepare the colums of the main experiment, confirm the the fact in a eppendorf in batch with a low volume of resin 20-40 ul, wash, add loading solution, and check that your protein was bounded. If yes, make the big purification follow the instructions of the supplier. Good luck

you have the right idea but the exchangers backwards. use deae or q if the protein is negatively charged (pH above pI) and s or cm if the protein is positively charged (pH below pI).


Im terribly sorry, I though that I was correct, but by other way I have the pleasure of be corrected by one of the most intelligent person of all the forums that I know. Thanks

-protolder-

protolder on Wed Dec 1 07:53:29 2010 said:


Im terribly sorry, I though that I was correct, but by other way I have the pleasure of be corrected by one of the most intelligent person of all the forums that I know. Thanks

it doesn't speak well for the forums you frequent.

-mdfenko-