PhosSTOP - 広範なホスファターゼに対して即座に効力を発揮

PhosSTOPカクテル錠は、ほぼすべての組織あるいは細胞(動物細胞・植物細胞・微生物や酵母を含む)からの抽出物に介在する、広範なホスファターゼに対して使用することができるタブレットです。毒性のないPhosSTOPカクテル錠は水に溶けやすく、有機溶媒(DMSOなど)を使用する必要がありません。



  • 完全な保護: 酸性およびアルカリ性ホスファターゼ・セリン/トレオニン ホスファターゼ(PP1, PP2AやPP2B等) ・チロシン ホスファターゼ(PTP)など、広範囲のホスファターゼ活性を阻害し、タンパク質を保護します ( Figure 1)。
  • 安全性:毒性のないPhosSTOP錠は水に溶けやすく、有機溶媒(DMSOなど)を使用する必要がありません。
  • 固定サンプルにも: 組織切片など、フォルマリン固定サンプル中のタンパク質のリン酸化状態も維持できます ( Figure 2)。
  • 利便性:適切なホスファターゼ インヒビターを調べるための手間暇が省けます。
  • かんたん:錠剤をバッファに加え、すみやかに溶解するだけの簡単手順です。
  • 長期保存: 冷蔵保存(+2~+8°C)した場合1カ月、冷凍保存(-15~-25°C)した場合は6カ月間安定しています。

包括的なタンパク質の保護のためにPhosSTOPと cOmplete プロテアーゼ インヒビターをセットでご使用ください。


PhosSTOPカクテル錠は、下記の広範なホスファターゼを阻害します。


  • 酸性ホスファターゼ
  • アルカリ性ホスファターゼ(e.g., PP1, PP2A and PP2B)
  • チロシン ホスファターゼ (PTP)
  • 2重特異性ホスファターゼ

PhosSTOPは、フォルマリンを含むバッファ中でも有効なので、パラフィン包埋組織切片にも使用することができます。包括的なタンパク質の保護のためにPhosSTOPと cOmplete プロテアーゼ インヒビターをセットでご使用ください。

 

Phosphatase Phosphatase Activity
(units/10 ml)
% Inhibition of Phosphatase Activity after 15 min Incubation
Calf alkaline phosphatase 140 U 98.4%
Potato acidic phosphatase 2 U 93.7%
Human acidic phosphatase 640 U 99.5%
Rabbit PP1 200 U 98.6%
Human PP2A  500 U 94.4%
Human PTP 500 U 96.7%

Table 1: Inhibition of phosphatase activity of isolated phosphatases.
PhosSTOP通常用量を添加、15分間のインキュベート後にアルカリ性(AP)および酸性ホスフォターゼ(SP) ・セリン/トレオニン ホスファターゼ(PP1とPP2A) ・チロシン ホスファターゼ(PTP)それぞれの阻害効果をMalachite Green アッセイにより検出した。



  AP SP PP1 PP2A 1 PTP 1
A 431 lysate 3 100% 88.5% 98.2% 80.2% 67.1% 1
COS lysate 3 100% 100% 95.8% 52.3% 1 68.1% 1
Maize extract 2 100% 69% 97.8% 72.2% 89.9%
Tobacco extract 2 93% 70.2% 96.8% 100% 96%
Insect cell lysate 3 100% 86.8% 94.6% 10.9% 1 50.2% 1

Table 2: Inhibition of phosphatase activity in different cell extracts.
サンプルに通常用量のライシス試薬とPhosSTOPを添加したのち、リン酸化ペプチドを添加した。遊離したリン酸はMalachite Green アッセイにより検出した。

1 Other enzymes in the cell extract may interfere with the assay results.
2 Lysis via P-PER Plant Protein Extraction Kit (Pierce Biotechnology, Inc.).
3 Lysis via Lysis M solution from cOmplete Lysis-M Kit  (Roche).


 

PhosSTOP - Western blot of an insect cell lysate.
Figure 1: Western blot of an insect cell lysate. Lanes 1 – 3: incubation without PhosSTOP Inhibitor for 0, 3, and 24 hours at +15°C to +25°C. Lanes 4 – 6: incubation with PhosSTOP for 0, 3, and 24 hours at +15°C to +25°C.
Development of western blot: blocking with 1x solution of Western Blocking Reagent in TBST, incubation with anti-phosphoserine/threonine antibody (BD Bioscience) in blocking solution. Incubation with secondary antibody (Anti Mouse-Ig, Chemicon) in blocking solution. Detection using LumiLightPLUS Western Blotting Substrate (Roche).




 

Detection of p44/42 MAP Kinase (pERK) on human ovarian cancer tissue after fixation for 24 hours in PBS containing 4% formalin (A) with 1x PhosSTOP or (B) without PhosSTOP. Figure 2: Detection of p44/42 MAP Kinase (pERK) on human ovarian cancer tissue after fixation for 24 hours in PBS containing 4% formalin (A) with 1x PhosSTOP or (B) without PhosSTOP. Tissue sections were stained with anti-pERK p44/42 MAPK (pERK) antibody (Cell Signaling Technology) and detected using EnVision+ System/HRP, Mouse (DAB+)(Dako).




 

Determination of bovine serum albumin (BSA) concentration </b>using the BCA assay (Pierce Biotechnology, Inc.) with and without addition of PhosSTOP.
Figure 3: Determination of bovine serum albumin (BSA) concentration using the BCA assay (Pierce Biotechnology, Inc.) with and without addition of PhosSTOP. The PhosSTOP shows almost no influence on BCA or Bradford protein assays.



 

The regulation of many biological processes and pathways is accomplished by the formation and cleavage of phosphate esters. The phosphorylation of proteins is carefully balanced by an interplay of protein kinases and phosphatases. To understand these pathways, a great deal of scientific work is currently being carried out within the pharmaceutical industry and other research institutes.
An important step to achieve this goal is to get an accurate view about the general, or specific phosphorylation status, which requires a preservation of the phosphorylation pattern. Phosphorylated protein can be dephosphorylated by either nonspecific phosphatase (e.g. alkaline phosphatase) or the more specific protein phosphatases like serine/threonine-specific, or tyrosine-specific phosphatases as well as dual-specificity phosphatases. Roche Applied Science has extensive experience with the isolation, purification, and analysis of many different proteins, as well as the best methods to protect these proteins from degradation by proteases, or from dephosphorylation by phosphatases.

Starting concentration: 1 tablet contains phosphatase inhibitors sufficient for 10 ml cell extract or buffer.
Specificity: Protects proteins against dephosphorylation. Inhibits phosphatase classes such as acid and alkaline phosphatases, as well as serine/threonine (PP1, PP2A, and PP2B) and tyrosine protein phosphatases (PTP) in bacterial, mammalian, yeast, and plant extracts.
Solubility/Stability: Soluble in aqueous buffers, or can be added directly to extraction media. The inhibitors are active in buffers containing formalin. Dissolve one tablet per 10 ml aqueous buffer. Alternatively, prepare a 10x stock solution in 1 ml water or 100 mM phosphate buffer, pH 7.0. Higher concentrations of stock solution are possible.
10x Stock solution is stable for 1 month at +2 to +8°C or at least 6 months at -15 to -25°C.
 

Quality

Performance tested with different isolated human phosphatases.


Read in this article:



 

Inhibition of Isolated Phosphatases


Phosphatases are ubiquitous. Depending on species, cell, or organ type and status of the particular cells used in the experiment, the spectrum and quantity of the different phosphatases vary significantly. Although each phosphatase class shows different substrate specificity, depending on the particular species, several isolated phosphatases have been tested in order to obtain some idea of the range of inhibition (see Table 1).


Phosphatases Units (U/10 ml) % Inhibition
Calf alkaline phosphatase (AP) 140 U 98.4%
Potato acidic phosphatase (SP) 2 U 93.7%
Human acidic phosphatase (SP) 640 U 99.5%
Rabbit PP1 200 U 98.6%
Human PP2A 500 U 94.4%
Human PTP 500 U 96.7%

Table 1. Effectively inhibit a broad range of phosphatases with a single PhosSTOP Tablet. After 15 minutes incubation (1 tablet per 10 ml), the inhibitory efficiency of PhosSTOP was evaluated for alkaline (AP) and acid (SP) phosphatases, as well as for serine/threonine (PP1 and PP2A), and tyrosine protein phosphatases (PTP). For each assay, a different phosphorylated peptide was used. Released phosphate was detected via a malachite green assay. The percentage given in the table is inhibitory efficiency. The final concentration of the dissolved PhosSTOP tablet in each assay was 1x.

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Inhibition of Phosphatases in Cell Lysates


Inhibition of phosphatases in mammalian cells


PhosSTOP Tablets deliver more effective phosphatase inhibition than other suppliers’ liquid phosphatase inhibitor cocktails in lysate from A431 human cancer cell line.

A431 cells were lysed using the Lysis-M Reagent from the cOmplete Lysis-M Kit (Roche Applied Science) according to the Instructions for Use. Lysates were treated with PhosSTOP Tablets or phosphatase inhibitor cocktails from other suppliers according to manufacturer’s instructions, and then assayed for activity of different phosphatases (see Figure 1).

For each phosphatase-specific assay, a corresponding phosphorylated peptide was added to the samples, and released phosphate was detected via a malachite green assay. The percentage given in the figure is inhibitory efficiency. The final concentration of the dissolved PhosSTOP Tablet in each assay was 1x.


Inhibition of phosphatase activity in lysate of A431 human cancer cells

Figure 1: Inhibition of phosphatase activity in lysate of A431 human cancer cells.
S1, S2, and C4 = Phosphatase Inhibitor Cocktails 1, 2, and 4 from other suppliers
AP and SP = Alkaline (AP) and Acid (SP) Phosphatases
PTP = Tyrosine Protein Phosphatases
PP1 and PP2A = Serine/Threonine Phosphatases

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Inhibition of phosphatases in insect cells


PhosSTOP protects proteins from phosphatases in insect cell lysates, as demonstrated on a western blot.

Insect cell lysates were treated with PhosSTOP or left untreated, and then incubated for the time periods indicated in Figure 2.


Western blot of an insect cell lysate

Figure 2: Western blot of an insect cell lysate. Detection via anti-phosphoserine antibodies. Development of western blot after blotting: blocking with 1x solution of Blocking Reagent (Roche Applied Science) in TBST and incubation with anti-phosphoserine/threonine antibody (BD Bioscience, 1:1000 dilution) in blocking solution. Incubation with secondary antibody (anti mouse-Ig, Chemicon, 1:1000 dilution) in blocking solution. Detection using Lumi-LightPLUS Western Blotting Substrate (Roche Applied Science).
Lanes 1 - 3: incubation without PhosSTOP for 0, 3, and 24 hours at +15°C to +25°C.
Lanes 4 - 6: incubation with PhosSTOP for 0, 3, and 24 hours at +15°C to +25°C.


PhosSTOP Tablets deliver more effective phosphatase inhibition than other suppliers’ liquid phosphatase inhibitor cocktails in lysate from an insect cell line.

Insect cells were lysed using the Lysis-M Reagent from the cOmplete Lysis-M Kit (Roche Applied Science) according to the Instructions for Use. Lysates were treated with PhosSTOP Tablets or phosphatase inhibitor cocktails from other suppliers according to manufacturer’s instructions, and then assayed for activity of different phosphatases (see Figure 3).

For each phosphatase-specific assay, a corresponding phosphorylated peptide was added to the samples, and released phosphate was detected via a malachite green assay. The percentage given in the figure is inhibitory efficiency. The final concentration of the dissolved PhosSTOP Tablet in each assay was 1x.


Inhibition of phosphatase activity in lysate from SF9 insect cells

Figure 3: Inhibition of phosphatase activity in lysate from SF9 insect cells.
S1 and S2 = Phosphatase Inhibitor Cocktails 1 and 2 from other suppliers
AP and SP = Alkaline (AP) and Acid (SP) Phosphatases
PTP = Tyrosine Protein Phosphatases
PP1, PP2A, and PP2B = Serine/Threonine Phosphatases

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Inhibition of phosphatases in plant cells


PhosSTOP Tablets deliver more effective phosphatase inhibition than other suppliers’ liquid phosphatase inhibitor cocktails in lysate from maize cells.
Maize cells (Zea mays) were lysed using the P-Per Plant Protein Extraction Kit (Pierce) according to pack insert instructions. Lysates were treated with PhosSTOP Tablets or phosphatase inhibitor cocktails from other suppliers according to manufacturer’s instructions, and then assayed for activity of different phosphatases (see Figure 4).
For each phosphatase-specific assay, a corresponding phosphorylated peptide was added to the samples, and released phosphate was detected via a malachite green assay. The percentage given in the figure is inhibitory efficiency. The final concentration of the dissolved PhosSTOP Tablet in each assay was 1x.


Inhibition of phosphatase activity in maize cell extract

Figure 4: Inhibition of phosphatase activity in maize cell extract.
S1, S2, and C4 = Phosphatase Inhibitor Cocktails 1, 2, and 4 from other suppliers
AP and SP = Alkaline (AP) and Acid (SP) Phosphatases
PTP = Tyrosine Protein Phosphatases
PP1 and PP2A = Serine/Threonine Phosphatases


PhosSTOP Tablets deliver more effective phosphatase inhibition than other suppliers’ liquid phosphatase inhibitor cocktails in lysate from tobacco cells.
Tobacco cells (Nicotiana x sanderae) were lysed using the P-Per Plant Protein Extraction Kit (Pierce) according to pack insert instructions. Lysates were treated with PhosSTOP Tablets or phosphatase inhibitor cocktails from other suppliers according to manufacturer’s instructions, and then assayed for activity of different phosphatases (see Figure 5).
For each phosphatase-specific assay, a corresponding phosphorylated peptide was added to the samples, and released phosphate was detected via a malachite green assay. The percentage given in the figure is inhibitory efficiency. The final concentration of the dissolved PhosSTOP Tablet in each assay was 1x.


Inhibition of phosphatase activity in tobacco cell extract

Figure 5: Inhibition of phosphatase activity in tobacco cell extract.
S1, S2, and C4 = Phosphatase Inhibitor Cocktails 1, 2, and 4 from other suppliers
AP and SP = Alkaline (AP) and Acid (SP) Phosphatases
PTP = Tyrosine Protein Phosphatases
PP1 and PP2A = Serine/Threonine Phosphatases

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Inhibition of Phosphatase in Tissue Sections


PhosSTOP Tablets can also be used to prevent dephosphorylation in formalin-fixed, paraffin-embedded (FFPE) tissue sections (see Figure 6).


Protection of proteins' phosphorylation state in formalin-fixed paraffin-embedded human ovarian cancer tissue with PhosSTOP Protection of proteins' phosphorylation state in formalin-fixed paraffin-embedded human ovarian cancer tissue without PhosSTOP

Figure 6: Protection of proteins' phosphorylation state in formalin-fixed paraffin-embedded human ovarian cancer tissue. Detection of pERK using p44/42 MAPK antibodies (cell signaling) on human ovarian cancer tissue after fixation with 4% buffered formalin solution with addition of PhosSTOP (Panel A) in comparison to no addition of PhosSTOP (Panel B). In addition, it was demonstrated that other non-phosphorylated markers could be detected equally well on formalin-fixed tissue sections with and without addition of PhosSTOP.

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Protein Assays



PhosSTOP inhibitors were evaluated for their effect on protein assays. The inhibitors show almost no influence on BCA (see Figure 7) or Bradford protein assays. However, we recommend running control experiments in order to ensure that no cooperative interference (tablet with components of the particular buffer) occurs.


Determination of bovine serum albumin (BSA) protein concentration using the BCA assay (Pierce) with and without addition of PhosSTOP


Figure 7: Determination of bovine serum albumin (BSA) protein concentration using the BCA assay (Pierce) with and without addition of PhosSTOP.
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