Protein phosphatase assay
This protocol describes the standard strategy for measuring Ser/Thr
protein phosphatase (PPase) activity in our laboratory using an artificial substrate (ex. Fzy-S50), a recombinant protein kinase (ex. Cdk) and [γ-32P]-ATP. This protocol can be modified/utilized to measure various PPase activity of your interest by changing substrate and kinase.
1, Purification of substrate protein 2, Phosphorylation of substrate 3, Phosphatase assay in Xenopus egg extract
1, Mochida and Hunt, 2007, Nature, 449(7160), 336-340, PMID 17882219 2, Mackintosh C. & Moorhead G. Protein phosphorylation: A practical
(ed. Hardie D.G.) pp 153-181 (Oxford University Press.1999)
Purification of substrate protein
This protocol uses an artificial substrate, which is composed of tag
protein (such as maltose binding protein) and a short peptide sequence derived from native phospho-protein.
Fuse 25a.a.-length phospho-peptide sequence to MBP or GST
expression vector. Phosphorylation site should be at the center of this peptide. I strongly recommend you to start with 5 or more different peptide sequences. Because some sequences are not expressed in bacteria or are not phosphorylated efficiently in vitro
. And I do NOT recommend putting more than 1 phosphorylation site in each substrate, as different sites could be dephosphorylated by different PPases.
BL21(DE3) strain + pLysS +pMALc2-FzyS50) Amylose resin (New England Biolab)
1 L of LB media supplemented with ampicil in & chloramphenicol Add 1 mM IPTG to your culture and incubate it for another 3 hrs @37 °C
Col ect bacterial cel s & wash cel pel et once in PBS Suspend them in 25 ml of Extraction buffer +lysozyme Freeze this solution using liquid nitrogen Thaw cel pellet and incubate it in 37°C water bath for 15 minutes Cool it down on ice Sonicate it for 20-30 seconds 3-4 times (until no viscosity) Centrifuge @10 krpm for 15 minutes @4 °C Apply the supernatant to 5 ml of amylose resin Rotate the mixture for 0.5 hr @4°C Wash the beads 3 times with the Extraction buffer Add 5 ml of Elution buffer to the beads and rotate for 10 minutes @R.T. Retrieve eluate Repeat elution twice more Concentrate 15 ml of eluate (5 ml x3) into 1-2 ml using a centrifugal filter unit Dialyze it 3 times against Elution buffer Measure protein concentration Stock it in 1-mg aliquots @-20°C You would get 10-20 mg substrate from 1L bacterial culture
r : 20 mM Tris-Cl pH7.5, 150 mM NaCl, 1 mM DTT, 1 mM PMSF,
2 mM Benzamidine, 1 mM EDTA, 1 mM EGTA, 0.1% Tween-20
: 20 mM Tris-HCl pH7.5, 150 mM NaCl, 10 mM maltose
Phosphorylation of substrate Materials
1 mg of substrate protein Active recombinant kinase [γ-32P]-ATP
Kinase (depends on how active your kinase is)
Incubate your reaction mixture at 37°C overnight (longer than 12 hrs) Add 200 µl of amylose resin (New England Biolab) to the reaction mix Rotate for 15 minutes Wash beads 5 times in Wash buffer Add 200 micro-L of Elution buffer to your beads Rotate for 10 minutes Retrieve 1st eluate to a new tube Repeat elution twice more, resulting in 600 micro-L of eluate Remove free ATP by using a filter unit and Wash buffer
(until radio activities of flowthrough does not change)
Concentrate substrate solution into 100 micro-L Measure its radioactivity using a liquid scintil ation counter If successful, your preparation would have 100 ~200 kcpm/micro-L Freeze in 10-20 micro-L aliquots @-20°C
5x CDK kinase buffer
: 0.1 M Hepes-Na pH7.8, 50 mM MgCl2, 75 mM KCl, 5
mM EGTA, 25 mM NaF, 0.1 M β-glycero PO4, 50 micro-M ATP, 5 mM DTT (Very low concentration of cold ATP is to get highly radio-active substrate)
: we use PerkinElmer NEG502A, 3000 Ci/mmol, 10 mCi/mL Wash buffer
: 20 mM Tris-Cl pH7.5, 150 mM NaCl, 0.05% Tween-20 Elution buffer
: Wash buffer +10 mM maltose If your [
γ-32P]-ATP is old
, do increase its amount used accordingly.
Phosphatase assay in Xenopus egg extract
egg extract (or your PPase source) Phosphorylated substrate
Reaction pre-mix for 10 assays
Okadaic acid* (100 micro-M in DMSO) or DMSO
To 10 micro-L Buffer (20 mM Tris-Cl pH7.5, 150 mM NaCl)
Mix egg extract (3 micro-L) and reaction pre-mix (1 micro-L) Incubate it @23°C for 4 minutes Add 20 micro-L of 10 % trichloroacetic acid to your reaction Vortex it for 5 seconds (you see white protein precipitate) Incubate it on ice for longer than 10 minutes (You can leave them on ice until al samples become ready) Centrifuge it for 5 minutes @13 krpm @4°C Retrieve al supernatant into a new tube, while trying not to take
Centrifuge it for another 10 minutes @13 krpm @4°C Retrieve 15 micro-L of its supernatant into a new tube Add 20 micro-L of 5% (w/v) ammonium molybdate** (in 0.5M H2SO4)
Add 80 micro-L of organic solvent mix*** (2-methyl-1-propanol : heptane = 50 : 50, water-saturated) Vortex it vigorously for 30 seconds Centrifuge it for 1 minute @13 krpm Take 50 micro-L of organic (upper) phase to a scintil ation tube with
appropriate scintil ant (we use Ecoscint A)
Shake it well by hand Measure radioactivity using a scintil ation counter
* 2.5 micro-M of okadaic acid can suppress most of PP2A activity in
egg extract. You need very high concentration of this kind of inhibitor, because, in crude cel extracts, PPases are expressed at concentrations much higher than their reported IC50 values.
** I use SIGMA #A7302, but products from other suppliers would work too. *** Molybdate/organic solvent extraction method is introduced in detail in
Mackintosh C. & Moorhead G. Protein phosphorylation: A practical approach
(ed. Hardie D. G.) pp 153-181 (Oxford University Press.1999).
It is crucial to keep protein concentration as high as in vivo
Because simple dilution of cel extract disrupt physiological regulation of PPases by unknown mechanism.
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