PKG Substrate | Cytiva思拓凡-Whatman滤纸滤膜滤器

PKG Substrate;

PKG Substrate 是一种 cGMP 依赖性的蛋白激酶 (PKG) 的选择性底物。

PKG Substrate Chemical Structure

规格	价格	是否有货	数量
1 mg	￥1100	In-stock
5 mg	￥3900	In-stock
10 mg	;	询价	;
50 mg	;	询价	;

* Please select Quantity before adding items.

生物活性

PKG Substrate is a selective substrate for cGMP-dependent protein kinase (PKG).

IC₅₀ Target

PKG^[1]

体外研究
(In Vitro)

Incorporation of [³³P]ATP into the synthetic peptide PKG substrate RKRSRAE is measured. N⁶-benzyl-ATP inhibits kinase activity of PKG Iα gatekeeper mutants but not WT. The serotonin transporter (SERT) is responsible for reuptake of serotonin (5-hydroxytryptamine) after its exocytotic release from neurons. SERT is regulated by several processes, including a cyclic GMP signaling pathway involving nitric oxide synthase, guanylyl cyclase, and PKG^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

分子量

902.01

Formula

C₃₅H₆₇N₁₇O₁₁

Sequence

Arg-Lys-Arg-Ser-Arg-Ala-Glu

Sequence Shortening

RKRSRAE

运输条件

Room temperature in continental US; may vary elsewhere.

储存方式

Protect from light, stored under nitrogen

Powder	-80deg;C	2 years
	-20deg;C	1 year

*In solvent : -80deg;C, 6 months; -20deg;C, 1 month (protect from light, stored under nitrogen)

Solvent Solubility

In Vitro:;

H₂O

Peptide Solubility and Storage Guidelines:

1.;;Calculate the length of the peptide.

2.;;Calculate the overall charge of the entire peptide according to the following table:

;	Contents	Assign value
Acidic amino acid	Asp (D), Glu (E), and the C-terminal -COOH.	-1
Basic amino acid	Arg (R), Lys (K), His (H), and the N-terminal -NH₂	+1
Neutral amino acid	Gly (G), Ala (A), Leu (L), Ile (I), Val (V), Cys (C), Met (M), Thr (T), Ser (S), Phe (F), Tyr (Y), Trp (W), Pro (P), Asn (N), Gln (Q)	0

3.;;Recommended solution:

Overall charge of peptide	Details
Negative (lt;0)	1.;;Try to dissolve the peptide in water first. 2.;;If water fails, add NH₄OH (lt;50 μL). 3.;;If the peptide still does not dissolve, add DMSO (50-100 μL) to solubilize the peptide.
Positive (gt;0)	1.;;Try to dissolve the peptide in water first. 2.;;If water fails, try dissolving the peptide in a 10%-30% acetic acid solution. 3.;;If the peptide still does not dissolve, try dissolving the peptide in a small amount of DMSO.
Zero (=0)	1.;;Try to dissolve the peptide in organic solvent (acetonitrile, methanol, etc.) first. 2.;;For very hydrophobic peptides, try dissolving the peptide in a small amount of DMSO, and then dilute the solution with water to the desired concentration.

参考文献

[1]. Wong A, et al. Cyclic GMP-dependent stimulation of serotonin transport does not involve direct transporter phosphorylation by cGMP-dependent protein kinase. J Biol Chem. 2012 Oct 19;287(43):36051-8.

Kinase Assay ^[1]	Kinase activity is measured by determining the amount of ³³P radioactivity incorporated from [³³P]ATP or [³³P]N⁶-benzyl-ATP into a PKG specific peptide substrate (RKRSRAE). The standard 75 μL assay mixture contains 0.15 μCi of [³³P]ATP, 10 μM ATP, 15 μM PKG peptide substrate, 2 μM PKI (a synthetic peptide inhibitor of cAMP-dependent protein kinase), 1 μg of purified kinase, and 100 μM 8-Br-cGMP in 50 mM HEPES buffer, pH 7.4, containing 10 mM MgCl₂, 0.1% Tween 20, and 1 mM DTT. After incubation at 30°C for 2 min, the reaction is immediately put on ice, and 20 μL of the assay mixture is spotted onto P81 phosphocellulose paper and then quenched in 0.42% H₃PO₄. The paper is further washed three times in 0.42% H₃PO₄ for 10 min with gentle agitation and rinsed once with acetone. After air drying, radioactivity on the paper is measured with a Beckman LS6500 liquid scintillation counter. For measuring the effect of N⁶-benzyl-ATP on the activity of PKG I utilizing ATP as a co-substrate, unlabeled N⁶-benzyl-ATP is added to each reaction at the indicated concentrations. Saturation kinetic analyses for K_m and V_max with ATP or N⁶-benzyl-ATP are performed over a concentration range (0.0015-100 μM) by adding unlabeled ATP or N⁶-benzyl-ATP to a given amount of [³³P]ATP or [³³P]N⁶-benzyl-ATP, respectively^[1]. MCE has not independently confirmed the accuracy of these methods. They are for reference only.
参考文献	[1]. Wong A, et al. Cyclic GMP-dependent stimulation of serotonin transport does not involve direct transporter phosphorylation by cGMP-dependent protein kinase. J Biol Chem. 2012 Oct 19;287(43):36051-8.

Kinase Assay
^[1]

Kinase activity is measured by determining the amount of ³³P radioactivity incorporated from [³³P]ATP or [³³P]N⁶-benzyl-ATP into a PKG specific peptide substrate (RKRSRAE). The standard 75 μL assay mixture contains 0.15 μCi of [³³P]ATP, 10 μM ATP, 15 μM PKG peptide substrate, 2 μM PKI (a synthetic peptide inhibitor of cAMP-dependent protein kinase), 1 μg of purified kinase, and 100 μM 8-Br-cGMP in 50 mM HEPES buffer, pH 7.4, containing 10 mM MgCl₂, 0.1% Tween 20, and 1 mM DTT. After incubation at 30°C for 2 min, the reaction is immediately put on ice, and 20 μL of the assay mixture is spotted onto P81 phosphocellulose paper and then quenched in 0.42% H₃PO₄. The paper is further washed three times in 0.42% H₃PO₄ for 10 min with gentle agitation and rinsed once with acetone. After air drying, radioactivity on the paper is measured with a Beckman LS6500 liquid scintillation counter. For measuring the effect of N⁶-benzyl-ATP on the activity of PKG I utilizing ATP as a co-substrate, unlabeled N⁶-benzyl-ATP is added to each reaction at the indicated concentrations. Saturation kinetic analyses for K_m and V_max with ATP or N⁶-benzyl-ATP are performed over a concentration range (0.0015-100 μM) by adding unlabeled ATP or N⁶-benzyl-ATP to a given amount of [³³P]ATP or [³³P]N⁶-benzyl-ATP, respectively^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

参考文献

[1]. Wong A, et al. Cyclic GMP-dependent stimulation of serotonin transport does not involve direct transporter phosphorylation by cGMP-dependent protein kinase. J Biol Chem. 2012 Oct 19;287(43):36051-8.