9 replies to this topic

[wenwen]

hi guys

does anyone has experiences about phosphorylating proteins?
for example, i want to phosphorylate the Ser77 of my protein
and i knew it can be phosphorylated by Akt kinase

can i co-express these two proteins and then purify the taget protein i want ?
assuming the purified protein should be phosphorylated and then checked by MALDI ?

or purifying the 2 proteins and then do in virto phosphorylation (supplied with ATP..) ?

any suggestions

thanks

wenwen

[knuf]

You can do it either way--it just depends on if there is anything else to your experiment which is more appropriate--but keep in mind that either way you do it, you're probably not going to get 100% phosphorylation of your protein. Additionally, you are likely going to get phosphorylation at sites in addition to your site of interest.

[wenwen]

hi
i had seen one paper said the kinase just specifically phosphorylates the site
and i think u are right i can not get 100 % phosphorylation of my protein
so i will need to seperate the WT and phosphorylated form but how ?

the phosphorylated protein should have 2 more negative charges from phosphate
it might cause conformation change so if  i run gel filtration
is it possible to see the different ? but the size is alomst the same
so ion exchange column might be more suitable ?  just found some cases they used strong ion exchange column to do seperation

any suggestion for the purification ?

wenwen

[ElHo]

Maybe 2D gel electrophoresis is an option for you. Different phosphorylations states usually result in distinct protein spots, which can be cut out and eluted.

[Inmost sun]

wenwen, on 06 January 2011 - 02:46 PM, said:

hi
i had seen one paper said the kinase just specifically phosphorylates the site
and i think u are right i can not get 100 % phosphorylation of my protein
so i will need to seperate the WT and phosphorylated form but how ?

the phosphorylated protein should have 2 more negative charges from phosphate
it might cause conformation change so if  i run gel filtration
is it possible to see the different ? but the size is alomst the same
so ion exchange column might be more suitable ?  just found some cases they used strong ion exchange column to do seperation

any suggestion for the purification ?

wenwen

you may separate phosphorylated from unphosphorylated forms by chromatofocusing; beware that you need relevant amounts of protein for biochromatography;

to guarantee site-specific phosphorylation by Akt, I would prefer to run phosphorylation in vitro, or you overexpress both proteins in a suitable cell system and try to trigger phosphorylation by cell stimulation

[knuf]

we have never tried to separate phosphorylated protein from nonphosphorylated protein. my suspicion is that even if under normal circumstances akt only phosphorylates the one site, under nonphysiological concentrations of  in vitro phosphorylation (or even in cells over expressing akt) you may get some nonphysiological phosphorylation--but this is just my suspicion. you could also do genetic pseudophosphorylation if its applicable--point mutate the site to an amino acid that endogenously has a negative charge (aspartic acid i believe is used most often) and in many cases (but not all) it will mimic the action of phosphorylation. we do this sometimes with success when we are interested in a specific site of phosphorylation, but obviously not if youre interested more in the kinase that is phosphorylating it.

Edited by kfunk106, 07 January 2011 - 07:15 AM.

[laurequillo]

wenwen, on 06 January 2011 - 02:46 PM, said:

hi
i had seen one paper said the kinase just specifically phosphorylates the site
and i think u are right i can not get 100 % phosphorylation of my protein
so i will need to seperate the WT and phosphorylated form but how ?

the phosphorylated protein should have 2 more negative charges from phosphate
it might cause conformation change so if  i run gel filtration
is it possible to see the different ? but the size is alomst the same
so ion exchange column might be more suitable ?  just found some cases they used strong ion exchange column to do seperation

any suggestion for the purification ?

wenwen

You can do a Mass Spectrometry approach, and using TiO2 beads you can separate the phosphorylated proteins from the non-phosphorylated ones.

Another option is to do a sequential Ip. First you overexpress both your proteins,your kinase and your substrate with a tag; you do a  Ip against the tag (Flag, Ha...), the you elute with a specific peptide (flag peptide, ha peptide) and do the second Ip against a specific or if you dont have it a pan-specific phospho (anti Serine, Threonine, Tyrosine) antibody, and you will have only the fraction of your protein which is phosphorylated

By the way, what do you want to see in your experiment? do you want to phosphorylate your protein, or just check the amount of protein phosphorylated? If you just want to phosphorylate your protein to see the efect or something like that,you can produce a phospho-mimic mutant in that serine, and you will have 100% of protein "phosphorylated".

Edited by laurequillo, 07 January 2011 - 07:36 AM.

[wenwen]

laurequillo, on 07 January 2011 - 07:27 AM, said:

wenwen, on 06 January 2011 - 02:46 PM, said:

hi
i had seen one paper said the kinase just specifically phosphorylates the site
and i think u are right i can not get 100 % phosphorylation of my protein
so i will need to seperate the WT and phosphorylated form but how ?

the phosphorylated protein should have 2 more negative charges from phosphate
it might cause conformation change so if  i run gel filtration
is it possible to see the different ? but the size is alomst the same
so ion exchange column might be more suitable ?  just found some cases they used strong ion exchange column to do seperation

any suggestion for the purification ?

wenwen

You can do a Mass Spectrometry approach, and using TiO2 beads you can separate the phosphorylated proteins from the non-phosphorylated ones.

Another option is to do a sequential Ip. First you overexpress both your proteins,your kinase and your substrate with a tag; you do a  Ip against the tag (Flag, Ha...), the you elute with a specific peptide (flag peptide, ha peptide) and do the second Ip against a specific or if you dont have it a pan-specific phospho (anti Serine, Threonine, Tyrosine) antibody, and you will have only the fraction of your protein which is phosphorylated

By the way, what do you want to see in your experiment? do you want to phosphorylate your protein, or just check the amount of protein phosphorylated? If you just want to phosphorylate your protein to see the efect or something like that,you can produce a phospho-mimic mutant in that serine, and you will have 100% of protein "phosphorylated".

hi  
my purpose for getting phosphorylated protein is protein crystallization
so i would need a bit protein for that
i had seen people did coexpression of kinase and substrate in the insect cell
but we donot have the equipment for insect cell and also getting "active" kinase
SIGMA sells active AKT
so i would choose to purify my substrate and do in vitro phosphorylation with the substrate
and then purify the phosphoryated substrate by ion exchange column ....

do u think it will work ?
wenwen

[laurequillo]

wenwen, on 09 January 2011 - 04:41 PM, said:

laurequillo, on 07 January 2011 - 07:27 AM, said:

wenwen, on 06 January 2011 - 02:46 PM, said:

hi
i had seen one paper said the kinase just specifically phosphorylates the site
and i think u are right i can not get 100 % phosphorylation of my protein
so i will need to seperate the WT and phosphorylated form but how ?

the phosphorylated protein should have 2 more negative charges from phosphate
it might cause conformation change so if  i run gel filtration
is it possible to see the different ? but the size is alomst the same
so ion exchange column might be more suitable ?  just found some cases they used strong ion exchange column to do seperation

any suggestion for the purification ?

wenwen

You can do a Mass Spectrometry approach, and using TiO2 beads you can separate the phosphorylated proteins from the non-phosphorylated ones.

Another option is to do a sequential Ip. First you overexpress both your proteins,your kinase and your substrate with a tag; you do a  Ip against the tag (Flag, Ha...), the you elute with a specific peptide (flag peptide, ha peptide) and do the second Ip against a specific or if you dont have it a pan-specific phospho (anti Serine, Threonine, Tyrosine) antibody, and you will have only the fraction of your protein which is phosphorylated

By the way, what do you want to see in your experiment? do you want to phosphorylate your protein, or just check the amount of protein phosphorylated? If you just want to phosphorylate your protein to see the efect or something like that,you can produce a phospho-mimic mutant in that serine, and you will have 100% of protein "phosphorylated".

hi  
my purpose for getting phosphorylated protein is protein crystallization
so i would need a bit protein for that
i had seen people did coexpression of kinase and substrate in the insect cell
but we donot have the equipment for insect cell and also getting "active" kinase
SIGMA sells active AKT
so i would choose to purify my substrate and do in vitro phosphorylation with the substrate
and then purify the phosphoryated substrate by ion exchange column ....

do u think it will work ?
wenwen

You can overexpress you kinase in cells,immunoprecipitate it in a buffer that allows kinase activity, and then phosphorylate in vitro your substrate

[knuf]

wenwen, on 09 January 2011 - 04:41 PM, said:

SIGMA sells active AKT
so i would choose to purify my substrate and do in vitro phosphorylation with the substrate
and then purify the phosphoryated substrate by ion exchange column ....

Yes, I think this is probably your best bet over expressing them in cells. You may want to check out this kit from Pierce. You may find it useful.
Ga-IDA Phosphopeptide Enrichment Kit