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GUIDEBOOK for Phos-tag™ Acrylamide AAL-107        
    for Phos-tag™ Acrylamide for the first trial ⇒ new pages

5 mM aqueous solution is now available!
manufacturer: Nard Institute


Effective Tool for Phosphorylated Protein Research!
◊ Operation Guidebook for Phos-tag™ SDS-PAGE ◊


  INDEX

    1. Phos-tag™ Acrylamide is ...
    2. Principle and Application
    3. Protocol
    4. Optimization of Phos-tag™ PAGE
    5. FAQ
    6. Related Products



Features:
♣ Phosphorylated proteins are easily separable using Phos-tag™ SDS-PAGE without any radioactive or chemical labels!
♣ Phosphorylated proteins are detectable corresponding to the degree of phosphorylation without any anti-phosphorylated antibodies!
♣ Applicable for the analysis of phosphorylation of endogenous proteins!



Description Wako Cat. No.
(Nard Institute #)
Package Size Applications
Phos-tag™ Acrylamide 300-93523
(AAL-107M)
2mg Separation of phosphorylated and
non-phosphorylated proteins using
SDS-PAGE with Phos-tag™ Acrylamide
304-93521
(AAL-107)
10mg
Phos-tag™ Acrylamide 5 mM Aqueous Solution
PDF PDF (1,242 KB/2 p)
304-93526
(AAL-107S1)
0.3 mL
(0.9 mg)
Ready-to-Use Solution for the first Phos-tag™ Acrylamide trial

Ready-to-Use Precast Phos-tag™ Acrylamide Gel
Description Wako Cat. No.
(Nard Institute #)
Package Size Applications
SuperSep™ Phos-tag™
 (50 μmol/L), 12.5 %, 13 well
195-16391 5 sheets Plate Size: 100 x 100 x 3 (mm)
Gel Size: 90 x 85 x 1 (mm)


1. Phos-tag™ Acrylamide is ...

Phos-tag™ Acrylamide can detect phosphorylated and non-phosphorylated proteins as different bands using Phos-tag™ SDS-PAGE without using radioisotopes. After separation, the gel can be utilized for western blotting and mass spectrometry!
Prepare Phos-tag™ SDS-PAGE by simply adding Phos-tag™ Acrylamide and MnCl2 to an acrylamide solution when preparing SDS-PAGE gels. During migration, the phosphorylated protein migrates with its phosphate group bound to the divalent metal ion in Phos-tag™ . This decreases the migration speed and phosphorylated/non-phosphorylated proteins are separated.
Suitable for kinase assay
◊ You can easily see the phosphorylation of your target proteins.
◊ You can see how much of the target protein is phosphorylated.
◊ You can determine the amount of phosphorylated/non-phosphorylated protein from the band intensity.

Almost the same basic mechanism as that of SDS-PAGE
◊ Simply add Phos-tag™ Acrylamide and MnCl2 to an acrylamide solution in the preparation of SDS-PAGE gel.
◊ Applicable to mass spectrometry
No need to prepare an antibody against the phosphorylated protein
◊ Simultaneous detection of phosphorylated/non-phosphorylated proteins by only using a total antibody
◊ Recognition of all phosphorylated forms of Ser/Thr/Tyr

Applicable for phosphorylation analysis of endogenous proteins
◊ Effect of stimulation on cells can be seen.
◊ Phosphorylation in KO mice, disease model mice, and pathological tissues can be analyzed.

Radioisotope labeling is not required.




2. Principle and Application


Phos-tag™ is a functional molecule that binds specifically phosphorylated ions. It is applicable for the specific separation of phosphorylated proteins (Phos-tag™ Acrylamide)
as well as for the detection using western blot (Phos-tag™ Biotin), purification (Phos-tag™ Agarose), and MALDI-TOF/MS (Phos-tag™ Mass Analytical Kit).

Product Purpose of use
Phos-tag™ Acrylamide Separation: Separation is possible by SDS-PAGE depending on the degree of phosphorylation.
Phos-tag™ Biotin Detection: A substitute for the anti-phospho antibody used in western blot.
Phos-tag™ Agarose Purification: Phosphorylated proteins are purified by column chromatography.
Phos-tag™ Mass Analytical Kit Analysis: This is used in MALDI-TOF/MS analysis to improve the detection sensitivity of phosphorylated molecules.
Features:
◊ Selectivity of binding of a phosphate ion (2-) is much higher than that of other anions.
◊ stable complex is formed under physiological conditions (pH 5 to 8).




Phosphorylated tyrosine was prepared by GST binding protein of tyrosine kinase Abl and the substrate peptide (Abltide) and separated with conventional SDS-PAGE and Phos-tag™ SDS-PAGE, respectively.

3. Protocol
[ I ] Phos-tag™ SDS-PAGE    Note: Always prepare the gel just before use.

(1) Preparation of reagents for Phos-tag™ SDS-PAGE
   Sol. A: 30 w/v% acrylamide solution (30% T, 3.3% C)
# acrylamide ···· 29.0 g
# N,N'-methylenebisacrylamide ···· 1.0 g
 ⇒ Prepare 100 mL solution by adding distilled water and filter the solution.
  ◊ Storage: Keep at 4 °Cin the dark
   Sol. B: 1.5 mol/L Tris/HCl buffered solution, pH 8.8 (x4 solution for resolving gel solution)
# Tris base (FW: 121, pKa = 8.2 at 20°C) ···· 18.2 g
# 6.0 mol/L HCl (0.19 equivalents of Tris) ···· 4.85 mL
 ⇒ Prepare the 100 mL solution by adding distilled water
  ◊ Storage: Keep at 4 °C.
   Sol. C: 0.50 mol/L Tris/HCl solution, pH 6.8 (x4 solution for stacking gel)
# Tris base ···· 6.06 g
# 6.0 mol/L HCl (0.96 equivalent of Tris base) ···· 8.0 mL
# distilled water ···· 90 mL
 ⇒ Adjust the pH to 6.8 using 6.0 mol/L HCl (ca. 0.1 mL), then prepare the 100 mL solution by adding distilled water
  ◊ Storage: Keep at 4 °C
  
Sol. D: 10% (w/v) SDS solution # SDS ···· 10.0 g
# distilled water ···· 90 mL
 ⇒ After stirring, prepare 100 mL solution by adding distilled water
  ◊ Storage: Keep at 4 °C
   Sol. E:5.0 mmol/L Phos-tag™ Acrylamide solution containing 3% (v/v) methanol

# Phos-tag™ AAL-107 ···· 10 mg   (2 mg)
# methanol ···· ···· ···· 0.10 mL  (0.02 mL)
# distilled water ···· 3.2 mL (0.64 mL)
* The amounts shown in parentheses are required for 2 mg of Phos-tag™ AAL-107.

The oily product, Phos-tag™ AAL-107 (10 mg), is provided in a small plastic tube and completely dissolved in 0.1 mL methanol. The solution should be diluted with 3.2 mL distilled water.

Note: If a trace amount of insoluble material appears as white fine powder (impurity) in the solution, it can be separated by centrifuging (2000 x g, 10 min.) using 2-mL microtubes.
Store the solution in 2-mL microtubes at 4 °C in the dark. From the supernatant solution, 45 mini-slab gels (50 μmol/L Phos-tag™ , 1 mm thick, 9 cm wide, 7.7-cm long) can be prepared.
   Sol. F: 10 mmol/L MnCl2* solution
*: Be careful not to confuse with magnesium.
# MnCl2·4H2O (FW: 198) ···· 0.10 g
# distilled water ···· 50 mL
Note: Do not use other anion salts such as Mn(NO3)2 or Mn(CH3COO)2 .
   Sol. G: 10% (w/v) diammonium peroxydisulfate solution
# (NH4)2S2O8 (FW: 228) ···· 10 mg
# distilled water ···· 0.10 mL
Note: Solution G should be freshly prepared just before acrylamide polymerization.
   Sol. H: Stock solution of running buffer, pH 8.3 (x10 solution)
  ◊ Storage: Keep at 4°C.
# Tris base (0.25 mol/L) ···· 15.1 g
# SDS ···· 5.0 g
# glycine (1.92 mol/L) ···· 72.0 g
 ⇒ Prepare 500 mL solution by adding distilled water. Avoid to adjust the pH by adding acid or base. Just before use, add 450 mL of distilled water to 50 mL of Solution H.
   Sol. I: Stock solution of Sample Buffer (x3 solution)
# Bromophenol Blue (BPB, a tracking dye) ···· 1.5 mg
# SDS ···· 0.60 g
# glycerol ···· 3.0 mL
# Sol. C: 0.50 mol/L Tris/HCl (pH 6.8) ···· 3.9 mL
# 2-mercaptoethanol ···· 1.5 mL
 ⇒ Prepare 10 mL solution by adding distilled water.
  ◊ Storage: Keep at -20°C
Usage of Sol. I: See "(2) Sample Preparation"

  
Sol. J: Acidic solution for fixation of proteins (1 L)
# acetic acid ···· 0.10 L
# methanol ···· 0.40 L
# distilled water ···· 0.50 L
   Sol. K: CBB staining solution (0.5 L)
# Coomassie Brilliant Blue (CBB) ···· 1.25 g
# methanol ···· 0.20 L
# acetic acid ···· 50 mL
# distilled water ···· 0.25 L
 ⇒ Dissolve CBB in methanol and add acetic acid and water.
   Sol. L: Washing and Destaining Solution (1 L)
# methanol ···· 0.25 L
# acetic acid ···· 0.10 L
# distilled water ···· 0.65 L
Note: When Quick CBB Plus is used as the Solution K, the solution L is not necessary because the destaining can be done in distilled water.
  
Resolving Gel Solution (0.375 mol/L Tris, 0.1 mmol/L MnCl2, 0.1% SDS)
(eg: total: 10 mL; acrylamide: 12 w/v%; Phos-tag™ AAL: 50 μmol/L)
* Refer to the following table with regard to other concentrations of acrylamide and Phos-tag™ .
# Sol. A: 30% (w/v) acrylamide solution ···· 4.00 mL
# Sol. B: 1.5 mol/L Tris/HCl solution, pH 8.8 ···· 2.50 mL
# Sol. E: 5.0 mmol/L Phos-tag™ AAL solution ···· 0.10 mL
# Sol. F: 10 mmol/L MnCl2 solution ···· 0.10 mL
# Sol. D: 10% (w/v) SDS solution ···· 0.10 mL
# TEMED (tetramethylethylenediamine) ···· 10 μL*
# Distilled water ···· 3.15 mL
  ~ Deaerate by stirring for 2 minutes ~
# Sol. G: 10% (w/v) diammonium peroxydisulfate solution ···· 50 μL*
*: The concentrations of TEMED and Solution G are example ones.
Please use each with an appropriate concentration you always prepare.

⇒ Reference: Preparation of 10 mL of resolving gel solution
Phos-tag™ conc. 20 μM 50 μM 100 μM
Acrylamide conc. 12% 10% 8% 6% 12% 10% 8% 6% 12% 10% 8% 6%
Sol. A (mL) 4 3.33 2.67 2 4 3.33 2.67 2 4 3.33 2.67 2
Sol. B (mL) 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
Sol. E (mL) 0.04 0.04 0.04 0.04 0.1 0.1 0.1 0.1 0.2 0.2 0.2 0.2
Sol. F (mL) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Sol. D (mL) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
TEMED (mL) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
distilled water (mL) 3.2 3.87 4.53 5.2 3.14 3.81 4.47 5.14 3.04 3.71 4.37 5.04
Sol. G (mL) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
Total (mL) 10 10 10 10 10 10 10 10 10 10 10 10
  
Stacking Gel Solution (0.125 mol/L Tris, 0.1% SDS)
(eg: in case of preparation of 10 mL solution with 4.5 w/v% acrylamide)
* In case of preparation of 2 mL solution, each volume is shown in parentheses.
# Sol. A: 30% (w/v) acrylamide solution ···· 1.50 mL (0.30 mL)
# Sol. C: 0.50 mol/L Tris/HCl solution, pH 6.8 ···· 2.50 mL (0.50 mL)
# Sol. D: 10% (w/v) SDS solution ···· 0.10 mL (20 μL)
# TEMED (tetramethylethylenediamine) ···· 10 μL (2 μL) *
# Distilled water ···· 5.84 mL (1.17 mL)
  ~ Deaerate by stirring for 2 minutes ~
# Sol. G: 10% (w/v) diammonium peroxydisulfate solution ···· 50 μL (10 μL) *
*: The concentrations of TEMED and Solution G are example ones. Please use each with an appropriate concentration you always prepare.
(2) Sample Preparation
1) Mix sample with 3 μL Sol. I and add an appropriate amount of distilled water to make 9 μL solution in a microcentrifuge tube. €Then, heat at 95°C for 5 minutes.
2) Allow the solution to cool to room temperature.
3) Load the sample solution (eg: 1.5 μL/well) using a micropipette.


(3) Electrophoresis
1) Assemble the electrophoresis equipments and fill the electrode chambers with the electrode buffer (= Sol. H)
2) Gently remove the comb from the stacking gel and load the sample into each well using a micropipette.
3) Attach the leads to the power supply. Run the gel under a constant current condition (30 mA/gel) until the BPB reaches€ the bottom of the resolving gel.

(4) CBB Staining
1) Fix the proteins in the gel by soaking in Sol. J (50 mL) for 10 minutes with gentle agitation.
2) Stain the gel by soaking in the staining solution (= 50 mL of Sol. I) for ca. 2 hours with gentle agitation.
3) Wash the gel in the destaining solution (= 50 mL of Sol. L, 3 times) to remove excess stain until the background is sufficiently clear.
4) Take a photograph of the gel.


[II] Tips for western blotting of Phos-tag™ SDS-PAGE gels
An additional procedure, ie, elimination of the manganese ion from the gel, is necessary before electroblotting. This procedure increases the transfer efficiency of the phosphorylated and non-phosphorylated proteins onto a PVDF membrane.

1) Just after electrophoresis, the gel is soaked in a general transfer buffer containing 1 mmol/L EDTA for a minimum of 10 minutes with gentle agitation.
2) Next, the gel is soaked in a general transfer buffer without EDTA for 10 minutes with gentle agitation
* a wet-tank method is strongly recommended for effective protein transfer from the Mn2+-Phos-tag ™ acrylamide gel to the
PVDF membrane. (The semi-dry method can also be used.)
* The blotting conditions, such as time and temperature, must be optimized for the phosphorylated target protein in the Phostag
™ gel.
* Change the temperature and treatment time with EDTA-buffer according to the gel thickness, etc. (eg: 1.5 mm thick: 20 minutes x twice).




[III] Tips for Mass Spectrometry of Phos-tag™ SDS-PAGE gels
No special procedures are necessary.


[IV] Trouble Shooting
Distortion of bands
The most common complaint for Phos-tag™ SDS-PAGE is "Distortion of bands." The following observations may be made:
(1) Prestained marker: Not only the lane of the marker but also the sample lane may be affected due to a difference in
the salt concentration among lanes.  ⇒ Prestained markers should not be used.

(2) Contamination: EDTA (Mn2+ is chelated), inorganic salts, surfactants, etc., cause distortion or tailing of bands. ⇒
Desalinate by precipitation with TCA or dialysis.

(3) Blank lanes: Blank lanes may cause distortion.  ⇒ Load the same amount of sample buffer in blank lanes.

Easy breaking of the gel
  With a protein of a high molecular weight, the gel is softened due to the low concentration of acrylamide.
  [Answer] Gels may be strengthened by increasing the ratio of N,N'-methylenebisacrylamide (eg: 24:1), addition of agarose (eg: 3% acrylamide + 0.5% agarose), and so on.

Trouble with transcription onto the membrane
  Treatment with EDTA may be insufficient. Increase the treatment time or frequency of exchange of the buffer containing EDTA
  (eg: 20 minutes x twice).
4. Optimization of Phos-tag™ PAGE Conditions
To obtain a high quality result using Phos-tag™ PAGE, optimization of the concentration of acrylamide and Phostag™ Acrylamide is essential.
Optimize the concentration of acrylamide first, followed by that of Phos-tag™ Acrylamide.

(1) Optimization of the concentration of acrylamide
First, identify the optimum concentration of acrylamide that allows migration of the target protein to the lowest end of the gel when conventional SDS-PAGE is used.

* In Phos-tag™ PAGE, the migration speed is slower than in conventional SDS-PAGE (including nonphosphorylated proteins) and, therefore, the concentration of acrylamide should be examined (see the below figure). The migration speed decreases as the concentration of Phos-tag™ increases.



(2) Identifying the appropriate concentration of Phos-tag™ Acrylamide

Then, identify the optimum concentration of Phos-tag™ Acrylamide.
Please evaluate the optimum concentration in the order of lowest to highest. (eg: 20 μM → 50 μM → 100 μM)

[ Cell Lysate ]
In case there is a large variety of proteins in your sample, eg: cell lysates, the concentration of Phos-tag™ should be 5 to 25 μM. However, a higher concentration, eg: 100 μM, is recommended in case of a lower concentration of the target protein, eg: non-overexpression systems.
* The optimum condition depends on the protein. Please find the appropriate condition setting for each target protein.


5. FAQs for Phos-tag™ Acrylamide

[ Determination ]
Q. Can phosphorylated proteins be assayed?
A. They can be assayed on the basis of the band intensity by using a quantitative staining (eg: CBB). A product such as "Quick-CBB PLUS" is recommended.
[Reference] Quick-CBB PLUS (1 L: Wako Cat. #178-00551; 250 mL: 174-00553)

[ Separation ]
Q. How large can a protein be (kDa) to be separated using this product?
A. A phosphorylated protein of 350 kDa has actually been separated (20 μM Phos-tag™ Acrylamide, 3% acrylamide + 0.5% agarose).
[Reference] Mobility shift detection of phosphorylation on large proteins using a Phos-tag SDS-PAGE gel strengthened with agarose, Proteomics, 9, 4098- 4101 (2009), E. Kinoshita, E. Kinoshita-Kikuta, H. Ujihara, and T. Koike

[ Staining ]
Q. In addition to CBB staining, is it possible to perform silver staining, fluorescent staining, and negative staining of the gel?
A. Yes.
[References]
   Silver Stain 2 Kit Wako (Wako Cat. #291-50301; 10 sheets)
   Silver Stain Kit Wako (Wako Cat. #299-13841; 10 sheets)
   Negative Gel Stain MS Kit (Wako Cat. #293-57701; 20 tests)

[ Preparation of reagents and gels ]
Q. What reagents and apparatuses do we need in addition to this product?
A. Manganese chloride (MnCl2) is needed (used as 10 mM solution). Other required reagents and apparatuses are common to those used for ordinary SDS-PAGE.

[ Use of Phos-tag™ Acrylamide ]
Q. How many tests can be performed with a 10-mg-package product?
A. It depends on the concentration of Phos-tag™ used. For example, about 100 plates at 20 μM, about 40 plates at 50 μM, and about 20 plates at 100 μM can be prepared from a 10-mg-package, if gels of 1-mm-thickness, 9-cm-width, and 7.7-cm-length are made.

[ Preparation of Phos-tag™ SDS-PAGE ]
Q. How is Phos-tag ™ SDS-PAGE prepared?
A. Simply add Phos-tag™ Acrylamide and manganese chloride (MnCl2) to an acrylamide solution when preparing gels for SDS-PAGE. Polymerization takes slightly more time than for SDS-PAGE. Since the prepared gel deteriorates within a few days, please prepare it just before use.

[ Stability of the prepared gel containing Phos-tag™ ]
Q. How long can the prepared gel containing Phos-tag™ Acrylamide be stored?
A. The gel deteriorates within a few days. Therefore, it should be prepared just before use.

[ Stability of the Phos-tag™ solution ]
Q. How long can the solutions in methanol and water be stored?
A. No remarkable decline in performance has been reported for 6 months by refrigeration under protection from light. The solutions seem to be storable for 1 year without any problem according to the doctors who are using the product. [ Preparation of the reagent ]
Q. I have experienced clouding of Phos-tag™ when I prepared a solution as described in the protocol. Is this normal?
A. Yes, it is. Clouding is attributed to methanol. The solution becomes clear after standing for a while.

Q. Does Phos-tag™ dissolve in water alone?
A. It is soluble in water, though it takes more time compared to dissolution in water containing methanol. If it does not dissolve completely, centrifuge the solution and use the supernatant.

[ Molecular marker ]
Q. What protein markers can we use?
A. A recombination product of the target protein is recommended. Prepare such marker protein using Escherichia coli, etc., and use it as a negative control for phosphorylation. It is used because the distance of migration in Phostag™ SDS-PAGE is not dependent on the molecular weight. A prestaining marker is not recommended because it affects other lanes as well. If a commercially available molecular weight marker is used, dissolve a powder product in sample buffer. However, it cannot be used as a molecular weight marker because Phos-tag™ SDS-PAGE is not dependent on the molecular weight. It is used simply to confirm proper functioning of Phos-tag™ SDS-PAGE from the distance of migration, or as a marker for the range of transfer onto a membrane in subsequent western blotting. A product suitable for use as protein marker is currently under investigation.

[ Phosphorylation reaction with coexisting ATP ]
Q. Does ATP in a phosphorylation reaction solution affect electrophoresis?

A. ATP had no particular effect at a concentration of 2.0 mM. The limit of use has not been investigated yet.

[ Precast gel ]
Q. Can we use the product in a precast gel by adding it to the sample solution?
A. No, you cannot.


6. Related Products

Related Products for Phos-tag™ SDS-PAGE and Western Blotting

Description Wako Cat. # Package Size Application
30 w/v% Acrylamide solution, 29:1 014-21705 500 mL Ready-to-use "Solution A"
10% SDS solution <Nippon Gene> 311-90271 100 mL Ready-to-use "Solution D"
313-90275 500 mL
Quick CBB Plus 174-00553 250 mL Ready-to-use "Solution K"
Fixing and destaining procedure are not required.
No organic solvents are necessary.
178-00551 1 L
Quick-CBB
◊ Staining solution A: 1 x 1L
◊ Staining solution B: 1 x 1L
299-50101 2 L By mixing staining solutions A and B, ready-touse "Solution K"
Silver Stain 2 Kit Wako 291-50301 10 sheets Highly sensitive detection of proteins and nucleic acids completed within an hour after separation with polyacrylamide gel electrophoresis.
Improved kit of Silver Stain Kit Wako
The sensitivity is 50 to 100 times higher than that of CBB stain!
Silver Stain Kit Wako 299-13841 10 sheets Highly sensitive detection completed within 100 min. after electrophoresis.
Negative Gel Stain MS Kit 293-57701 20 tests Since protein bands remain clear, applicable to the subsequent studies of protein such as sequencing and mass analysis of peptide
Alkaline Phosphatase
<for Biochemistry>
018-10693 50 U Applicable for dephosphorylation of protein samples
012-10691 100 U
Easy Separator 058-07681 1 set Polyacrylamide gel electrophoresis apparatus for Super-Sep
ImmunoStar LD
◊ Luminescence solution A
◊ Luminescence solution B
296-69901 200 cm2 Highly sensitive (femto gram level) immunoblotting, utilizing detection by enhanced chemiluminescence using a unique luminol derivative L-012 as substrate.
Not available for sale in the US and Europe.
292-69903 1,000 cm2
290-69904 2,000 cm2
Immuno Enhancer 294-68601 2 assays Ready-to-Use Immunoreaction Enhancer for western blotting and ELISA
290-68603 10 assays
298-68604 40 assays
Other Phos-tag™ Products
Description Wako Cat. # Package Size Applications
Phos-tag™ Agarose 302-93561 0.5 mL Purification of phosphorylated proteins using column chromatography
308-93563 3 mL
Phos-tag™ Mass Analytical Kit 305-93551 1 set Analysis by MALDI-TOF/MS
Phos-tag™ Biotin BLT-104 301-93531 10 mg This is used as an alternative to an antiphospho antibody. It is recommended when use of an anti-serine/threonine phospho antibody is difficult.
Phos-tag™ Biotin BLT-105 308-93541 10 mg
Phos-tag™ Biotin BLT-111
1 mL Aqueous Solution
308-97201 0.1 mL This is high sensitive Phos-tag™ Biotin BTL-104.

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