research phos-tag™ tool kit - alpha laboratories · affinity for the phosphate dianion, po. 4 2-,...

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Phos-tag™ Tool KitProtein phosphorylation analysis - radiation free

Introducing Phos-tag 2

Phos-tag Acrylamide 3

Phos-tag Biotin 8

Phos-tag Agarose 9

Phos-tag Mass Analytical Kit 10

Phos-tag Protocols 11

Solutions 18

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Introducing Phos-tag™Phos-tagTM is a novel labelling system for analysis of phosphorylation. The Phos-tag, developed by the Department of Functional Molecular Science at Hiroshima University, is a functional molecule that binds phosphorylated ions. Under physiological conditions pH 5-8, the Phos-tag forms a stable complex with anionic substituents, particularly phosphoric esters with a single ester bond. Affinity for the phosphate dianion, PO4

2-, is much higher than other negative ions making binding selective for phosphate groups.

Phos-tag can therefore take the place of conventional enzyme immunoassay and radioactive isotope methods as an agent for capturing substances with a phosphate monoester group in phosphorylation analysis techniques.

Protein phosphorylation is a post-translation modification in which a serine, threonine or tyrosine residue is phosphorylated by a protein kinase. Phosphorylation is the most common form of reversible post-translation modification and it is estimated that up to 30% of cellular proteins are phosphorylated at any time. Dephosphorylation to remove a phosphate group reverses the process and the phosphorylation-dephosphorylation reaction is important for regulating protein function.

Phosphoregulation is vital for many signal transduction pathways and controls numerous cellular processes including metabolism, gene transcription, cytoskeletal arrangement, cell-cycle progression, protein stability and apoptosis. Phos-tag technology can be used in a number of innovative methods for separation, purification, detection and analysis of protein phosphorylation to aid the investigation of regulatory pathways.

Discover the power of Phos-tag

Product Purpose of UsePhos-tag™ Acrylamide Separation: Separation is possible by SDS-PAGE depending on the degree of phosphorylation

SuperSep Phos-tag™ Separation: Ready-to-use Pre-cast gel containing 50µM Phos-tag™ Acrylamide

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

Introduction

Basic Phos-tag Structure M2+=Mn2+ or Zn2+

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Phos-tag™ AcrylamidePhos-tag Acrylamide is an acrylamide-pendant molecule that can be used for Mobility Shift Detection of Phosphorylated Proteins using phosphate affinity SDS-PAGE. The method requires a general mini-slab PAGE system and avoids the use of radio isotopes. The functioning Phos-tag molecule contains two divalent metal ions and both manganese and zinc ion Phos-tag SDS-PAGE techniques have been described by researchers at Hiroshima University.

Phos-tag Acrylamide and manganese chloride/zinc chloride are incorporated into the SDS-PAGE resolving gel, the Phos-tag then binds the phosphate groups of proteins in the sample. The bound Phos-tag decreases the migration speed of the phosphorylated protein enabling separation of phosphorylated and non-phosphorylated forms. Phos-tag SDS PAGE can be followed with further downstream analysis such as Western blotting or MALDI-TOF MS.

Phosphate affinity SDS-PAGE PrinciplePhosphorylated proteins are bound by Phos-tag as they migrate through the gel. This reduces the migration speed of the phosphorylated protein forms enabling separation. Distinct phosphoprotein isotypes with different positions or numbers of phosphorylation can also be separated depending upon the degree of phosphorylation.

Optimisation for Phos-tag SDS PAGEWe recommend that both acrylamide and Phos-tag concentrations be optimised for the target protein of interest to obtain the best results. When using Phos-tag SDS PAGE the migration speed of both phosphorylated and non-phosphorylated proteins is slower than conventional SDS-PAGE. The migration speed decreases as the Phos-tag concentration increases.

To begin with it is necessary to optimise the acrylamide concentration. The relative mobility (Rf) relates to the protein of interest and is calculated with reference to a tracking dye or marker protein: Rf = distance migrated by protein distance migrated by dye

Run a conventional SDS-PAGE gel until the Bromophenol Blue (BPB) from the sample buffer reaches the end of the resolving gel. The position of the BPB is defined as Rf 1. Adjust the acrylamide concentration of gel until the target protein is Rf 0.8 or 0.9. This acrylamide percentage should be optimal for Phos-tag SDS PAGE. As a starting point proteins greater than 60kDa are typically ~6% and less that 60kDa ~8%.

Phos-tag™ Acrylamide

Phos-tag Acrylamide AAL-107 MW 595

Phos-tagTM

SDS-PAGE SDS-PAGE

Protein band

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Handy Hint: Gels with a low acrylamide percentage are friable and difficult to handle post

electrophoresis when staining or blotting. One way to address the problem of gel breakage is to increase the relative amount of methylenebisacrylamide to acrylamide, for example 24:1.

In the case of high molecular weight proteins where gels may contain less that 4% acrylamide the gel strength can be increased by adding agarose. Kinoshita et al, 2009 reported separation of a 350kDa protein using a 3% acrylamide and 0.5% agarose gel.

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RRWhen optimising the Phos-tag concentration start with the lowest concentration and work up. Typically the suggested concentrations for evaluations would be 20µM, 50µM and 100µM.

In general, a higher concentration will give a greater separation capacity. An example of this seen below is α-casein, better separation is observed at 100µM Phos-tag than 50µM Phos-tag (10% polyacrylamide, Coomassie Brilliant Blue (CBB) stained gel).

However, increasing concentrations of Phos-tag will reduce migration velocity and in some instances a lower concentation will be optimal. This is demonstrated by ovalbumin where greater separation is see at 50µM than 100µM or 150µM (7.5% polyacrylamide, CBB stained gel).

Handy Hint: In cases where there is a large variety of proteins in your sample, e.g.

cell lysates, the Phos-tag concentration should be 5-25µM. In cases of lower concentrations of the target protein e.g. non-overexpression system a high concentration of Phos-tag such as 100µM is suggested.

[Information provided by Y Sugiyama at Science Research Centre, Kochi University]

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Phos-tag™ SDS-PAGE Applications: Downstream AnalysisFollowing Phos-tag SDS-PAGE further analysis techniques can also be applied. Phos-tag SDS-PAGE gels have been used with a number of gel staining techniques including Coomassie Brilliant Blue, silver stain, negative stain and fluorescent staining.

Phos-tag SDS-PAGE gels can also be blotted for immunodetection or proteins recovered from the gel for analysis by mass spectroscopy.

The power of Phos-tag can even be incorporated into 2D electrophoresis, use as the second dimension after IEF.

Handy Hint: High background staining can be prevented by eliminating metal

ions in the gel with EDTA pre-treatment.

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Gel-staining

Western blotting

Mass Analysis

2D Electrophoresis

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Phos-tag SDS-PAGE Applications: Kinase and Phosphatase Assays

Phos-tag SDS-PAGE Applications: Western BlottingPhos-tag gels can also be used for Western blotting. The separation of proteins can enable detection of phosphorylated target protein with a general antibody, instead of probing twice with both anti-phosphorylated and anti-non-phosphorylated antibodies. For Western blotting the use of a PVDF membrane is recommended and it is important to pre-treat the gel with EDTA to eliminate ions (Mn2+ or Zn2+) as this increases the transfer efficiency.

After electrophoresis the gel is soaked in transfer or running buffer containing 1-10mmol EDTA with agitation for a minimum of 10 min to chelate the metal ions in the gel. This step can repeated twice more if desired to flush out the ions and may be adapted according to the gel thickness, for example a 1.5mm gel might be treated twice with 20 min incubations.

The gel is then washed in buffer (transfer or running as used for EDTA treatment) to remove the EDTA before transfer. A wet-tank transfer is recommended but semi-dry methods may also be used. It is important to optimise the transfer conditions, i.e. time and temperature, for your phosphorylated protein target.

Handy Hint: ATP at 2.0mM in a phosphorylation reaction solutions does not affect Phos-tag SDS-PAGE.

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Handy Hint: Pre-stained molecular markers can cause distortion of the bands. Whilst

they do not reflect molecular weight they can be useful to give an index of transfer efficiency we recommend leaving an empty lane between these and the sample if you are using them to minimise band distortion. Instead of using MW Markers as a negative control for phosphorylation we recommend using alkaline phosphatase treated sample or recombinant target protein.

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Membranes after transferSample: WIDE-VIEW™III Prestain Marker(Code No. 230-02461)*

No EDTA processing 1mM EDTA for 10 minutes x 2 10mM EDTA for 10 minutes x 1 1mM EDTA for 10 minutes x 2

EDTA solution is prepared with 1x running buffer. 12.5% Phos-tag™ Super Sep (50mM)

Marked transfer efficiency drop occurs without EDTA processing. For a reliable transfer, 1-10mM EDTA process for 10 minutes x 2 is recommended. * Molecular weight is not reflected in Phos-tag™ gel. Use the transfer efficiency as a standard.

Phosphorylated + Non-phosphorylated

Phosphorylated

Non-phosphorylated

Abl tyrosine kinase assay:Phosphorylation of Ablitide-GST by Abl kinase on (a) standard and (b) Mn2+-Phos-tag SDS-PAGE, stained with CBB.

(a)

(b)

Kinase Reaction Time (min)0 1 5 10 20 30 60

Phosphorylated + ß-casein

Non-phosphorylated ß-casein

Alkaline phosphatase assay: Dephosphorylation of ß-casein by alkaline phosphatase on (c) Mn2+-Phos-tag SDS-PAGE, stained with CBB.

0 1 5 10 20 30 60

(c)

Phosphatase Reaction Time (min)

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Phos-tag SDS-PAGE Applications: Cdk-5-activated p35 PhosphorylationPhos-tag SDS-PAGE and Western blotting were used by Professor Tomohisa Hosokawa at the Brain Science Institute, RIKEN to investigate the phosphorylation site of cyclin-dependent kinase 5 (Cdk-5)-activated sub-unit p35. Ser8 and Thr138 are known major sites of p35 phosphorylation and the following Ala substitution mutants have been produced S8A – substitution of Ser8; T138A – Substitution at Thr138 and 2A: substitution at both Ser8 and Thr138. The group evaluated p35 phosphorylation by co-expressing these mutants with Cdk-5 or kinase negative cyclin-dependant kinase-5(knCdk-5) in COS-7 cells (green monkey fibroblast cell line).

The resulting cell lysates were then run using Phos-tag SDS-PAGE and blotted before probing with Anti-p35 antibody. The clear mobility shift enabled differentiation between phosphorylated and non-phosphorylated proteins using just one antibody.

Phos-tag SDS-PAGE Applications: Second stage 2D ElectrophoresisPhos-tag SDS-PAGE can also be incorporated into 2D electrophoresis applications as demonstrated by Professor Yayoi Kimura Yokohama City University. Phos-tag SDS PAGE was used as the second dimension of separation when studying phosphorylated isoforms of heterogeneous ribonucleoprotein K (hnRNP K). Cultured 2774.1 cells (mouse macrophage cell line) were stimulated with LPS, hnRNP K was susbsequently separated from a nuclear homogenate using immnoprecipitation. To separate hnRNP K isoforms, the extract was run using an IPG strip gel (pH 4.37-5.9) in the first dimension and then Phos-tag SDS-PAGE in the second dimension (25µM Phos-tag Acrylamide, 7.5% polyacrylamide gel). Isoforms and modification sites of the various spots were subsequently identified using a mass spectrometer.

From lanes 1 (L2, L4) and 5 (M1): p35 is phosphorylated, depending on Cdk-5.From lanes 1 (L2, L4) and 3 (L2, L4): With about half of p35, Thr138 is phosphorylated at kinase-negative Cdk-5, and Thr138 is also phosphorylated by kinase other than Cdk-5. From lanes 5 (M1) and 6 (L3, L4): Ser8 and Thr138 are main phosphorylation sites. From lanes 5 (M1), 7 (L1, L2) and 8 (M2): M1 is the phosphorylation site for Ser8 and Thr138. M2 is the phosphorylation site for Ser8 only. L1 and L2 are the phosphorylation sites for Thr138 only.※ X in L1, L3: not yet identified※ L4: non-phosphorylated p35

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Acidic 1st: IPG Basic

25µM Phos-tagTM Acrylamide, 75% polyacrylamide gel

Phosphorylated forms

p-Ser116/p-Ser284 (spots 1,2) p-Ser116 (spots 3,4,5,6)

p-Ser284 (spots 7,8)

Non-phosphorylated forms (spots 9,10,11,12)

※ Each isoform originates in a splicing variant. Isoform 1,3:C termination: SGKFF Isoform 2,4:C termination: ADVEGF Isoform 3,4:1 exon short

Each phosphorylated form was distinguished at the same isoelectric point, respectively. (eg: spots 6 vs. 8 and spots 4 vs. 7)

Handy Hint: No EDTA treatment of the gel is required for down-stream mass analysis.

Just follow the usual procedure for in-gel digestion after SDS-PAGE.

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The Phos-tag Acrylamide RangePhos-tag Acrylamide can be used to produce gels in various concentrations and different sizes. Therefore the number of gels that can be produced per pack will vary. As a general guide when casting 1mm thick gels 9cm (w) x 7.7cm (l) a 10mg vial of Acrylamide is sufficient for ~100 gels at 20µM, ~ 40 gels at 50µM or ~ 20 gels at 100µM.

SuperSep Phos-tag™ Pre-cast Polyacrylamide GelsThe convenience of a ready made gel. The Phos-tag SuperSep range of pre-cast gels are produced with a 50µmol/L Phos-tag Acrylamide and use zinc as the supplemental metal. Both 12.5% and 15% acrylamide gels are available with a choice of 13 or 17 wells. If you are using a molecular weight marker we recommend WIDE-VIEW Pre-stained marker for minimal band distortion.

The Phos-tag Acrylamide Range

Cat. No. Description Pack Size304-93526 Phos-tag Acrylamide AAL-107 5mM 0.3ml

300-93523 Phos-Tag Acrylamide AAL-107 2mg

304-93521 Phos-Tag Acrylamide AAL-107 10mg

Phos-tag™ 20µM 50µM 100µM0.3ml Aqueous Solution (0.9mg)

~ 9 ~ 4 ~ 2

2mg package ~ 20 ~ 8 ~ 4

10mg package ~ 100 ~ 4 ~ 20

Handy Hint: Phos-tag Acrylamide gels will deteriorate within a few days of casting.

For best results prepare your gels directly before use.

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M 1 2 3 4 5

Phosphorylatedß-casein +dephosphorylated ß-casein

M 1 2 3 4 5

Phosphorylated ß-casein

dephosphorylated ß-casein

The SuperSep Pre-cast Gel Range

Cat. No. Description Pack Size195-16391 Phos-tag SuperSep 12.5% 13 Well 5

193-16571 Phos-tag SuperSep 12.5% 17 Well 5

193-16691 Phos-tag SuperSep 15% 13 Well 5

196-16701 Phos-tag SuperSep 15% 17 Well 5

199-14971 SuperSep Ace 12.5% 13 Well 10

196-14981 SuperSep Ace 12.5% 17 Well 10

193-14991 SuperSep Ace 15% 13 Well 10

190-15001 SuperSep Ace 15% 17 Well 10

230-02461 WIDE-VIEW III Pre-stained Protein Marker 500µl

(See left) A time course of ß-casein phosphorylation shown on 12.5% SuperSep ACE and 12.5% SuperSep Phos-tag gels.

M: WIDE-VIEW™ Prestained Protein Size Marker III

1 : 0 min. Albumin (with AP treatment)2 : 15 min. Albumin (with AP treatment)3 : 30 min. ß-casein (with AP treatment)4 : 45 min. ß-casein (with AP treatment)5 : 60 min. ß-casein (with AP treatment)

SuperSep ACE SuperSep Phos-tag

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Phos-tag™ Biotin

Phos-tag™ BiotinPhos-tag Biotin ligands can be used to detect phosphorylated proteins on a PVDF membrane; use as an alternative to anti-phosphorylated antibodies to probe a Western blot. The functioning Phos-tag molecule contains two divalent metal ions and biotinylated Phos-tag Zn2+ ligands can be used to detect phosphorylated serine, threonine and tyrosine residues. The method requires streptavadin-conjugated horseradish peroxidase and chemiluminescent detection reagents.

There are three Phos-tag Biotin ligands: BTL-104, BTL-105 and BTL-111 which contain linkers with different lengths. BTL-104 and BTL-111 are preferred for Western blot analysis. BTL-104 has high solubility but the long hydrophyllic spacer gives BTL-111 higher sensitivity in this method.

Unlike traditional immunodetection of proteins there is no need to block the membrane before probing. Once a blot has been probed with Phos-tag Biotin it can be stripped and subsequently re-probed. The use of PVDF membrane is recommended when using Phos-tag Biotin

Phos-tag Biotin Western Blotting PrincipleThe Phos-tag Biotin ligand binds phophorylated amino acids captured on the PVDF membrane. HRP streptavadin conjugate is then applied which binds to the Biotin. A luminescent substrate is added enabling chemiluminescent detection of phosphorylated targets.

■ No need to block before probing ■ Sensitivity at nanogram level■ Strip and re-probe blot using conventional immunoprotocols

Phos-tag Biotin Application: Detection of Phosphoproteins by Western BlotDephosphorylation of ß–casein by alkaline phosphatase: Western blots were probed using BTL-111 and BTL-104.

The Phos-tag Biotin Range

Cat. No. Description Pack Size301-93531 Phos-Tag Biotin BTL-104 10mg

308-93541 Phos-Tag Biotin BTL-105 10mg

308-97201 Phos-tag Biotin BTL-111 0.1ml

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Phos-tag™ Agarose

Phos-tag™ AgarosePhos-tag™ Agarose can be used for separation, purification and enrichment of phosphorylated substances. Based on immobilised metal affinity chromatography (IMAC) the method uses a phosphate-binding tag molecule (a dinuclear zinc(II) complex) attached to highly cross-linked agarose (Phos-tag™ Agarose). The Phos-tag Agarose is used to construct a chromatography column which binds phosphorylated proteins within the sample, these are then eluted and can be subjected to down-stream analysis by mass spectrometry or SDS-PAGE electrophoresis.

Phosphate affinity chromatography Principle

Phosphate affinity chromatography Application: Enrichment of phosphoproteins

Lysate was applied on a column filled with Phos-tag Agarose. Phosphorylated proteins were concentrated in absorbed fraction.

Purification of phosphorylated proteins in A431 lysateM: Molecular MarkerLane 1: Non absorbed fractionLane 2: Absorbed fractionLane 3: Column rinsing fraction

(Left) Fluorescence staining(Right) Western blotting with anti-phosphorylated Tyr.

M 1 2 3 M 1 2 3

Handy Hint: The use of elution buffers with a high salt content can be problematic

for down-stream SDS-PAGE analysis, resulting in distorted bands. To avoid the hassle of desalting your samples elute with SDS-PAGE sample buffer instead.

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The Phos-tag Agarose Range

Cat. No. Description Pack Size302-93561 Phos-Tag Agarose AG-501 0.5ml

308-93563 Phos-Tag Agarose AG-503 3ml

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Phos-tag™ Mass Analytical Kit

Phos-tag™ Mass Analytical KitThe Phos-tag™ Mass Analytical Kit can be used to provide a simple, rapid and sensitive matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) analysis of phosphorylated compounds (RO-PO3

2-). The use of the Phos-tag complex can increase the sensitivity of detection by MALDI-TOF for phopholipids and phosphopeptides in positive mode. Samples are mixed with matrix and an isotopic Zn2+-Phos-tag complex prior to MALDI-TOF analysis. Phenolic and amine matrices are recommend and should be prepared in an appropriate organic solvent such as ethanol or acetonitrile.

MALDI-TOF MS with Zn2+- Phos-tag Prinicple

Kit contents:

Phos-tag MS-101L 5mg

Phos-tag MS-101H 5mg

Phos-tag MS-101N 10mg

Phos-tag MS-101N contains natural zinc and therefore a mixture of isotopes, we recommend using this for initial optimisation. The 64Zn and 68Zn istotopes can then be used to verify the presence of phosphate groups. Measurement of a single sample with these reagents therefore results in a difference in m/z of 16.

Phos-tag MS-101N has also been used in ESI MS analysis as described by Prudent et al, 2008.

Handy Hint: High background staining can be prevented by eliminating metal

ions in the gel with EDTA pre-treatment.

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The Phos-tag Mass Analytical Kit

Cat. No. Description Pack Size305-93551 Phos-tag Mass Analytical Kit Each

Handy Hint: Following Phos-tag SDS-PAGE isolation there is no need to remove the

Phos-tag before the in-gel digestion.

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Phos-tag™ Protocols

Phos-tag™ ProtocolsPhos-tag was developed by the Department of Functional Molecular Science at Hiroshima University who have generated a number of protocols illustrating the use of Phos-tag products, including methods for SDS-PAGE, Western blotting, Column chromatography and MALDI-TOF MS analysis.

Phosphate affinity SDS-PAGE Protocols E. Kinoshita-Kikuta, E. Kinoshita & T. Koike (Hiroshima University) Ver 11 (2013/6) http://home.hiroshima-u.ac.jp/tkoike/pro/PT_PAGE_e.pdf

Preparing Mn2+ Phos-tag SDS-PAGE Gels

1. Set up the casting apparatus (e.g. a mini-slab gel system, 1mm-thick).

2. Prepare the resolving gel solution:

Resolving Gel Solution (10 mL: 12% (w/v) Acrylamide and 50 µmol/L Phos-tag AAL)* *Remember to optimise acrylamide and Phos-tag concentrations for each target protein

30% (w/v) Acrylamide Solution 4.00 mL1.5 mol/L Tris/HCl Solution, pH 8.8 2.50 mL 5.0 mmol/L Phos-tag AAL Solution 0.10 mL 10 mmol/L MnCl2 Solution 0.10 mL 10% (w/v) SDS Solution 0.10 mL TEMED (tetramethylethylenediamine) 10 µL Distilled Water 3.15 mL

Mix reagents and degas for 2 min whilst stirring

3. Add 20-50µl of 10% Ammonium Persulphate Solution to the degassed solution and stir gently to mix.

4. Pour the resolving gel solution between the glass plates and finish by topping the gel with a layer of butanol-saturated water. Allow the gel solution to polymerise for 1 h at room temperature.

5. Prepare the stacking gel solution:

Stacking Gel Solution (10ml: 4.5% (w/v) Acrylamide)

Mix reagents and degas for 2 min whilst stirring:

30% (w/v) Acrylamide Solution 1.50 mL 0.50 mol/L Tris/HCl Solution, pH 6.8 2.50 mL 10% (w/v) SDS Solution 0.10 mL TEMED (tetramethylethylenediamine) 10 µL Distilled Water 5.84 mL Add 20-50µl of 10% (w/v) Ammonium Persulphate Solution and mix gently.

6. Rinse the top of the resolving gel with distilled water and remove the residual liquid with a paper towel.

7. Pour the stacking gel solution on top of the resolving gel and then insert a comb.

8. Allow the gel solution to polymerise for 1 h at room temperature.

Handy Hint: Recipes for solutions used in Phos-tag SDS-PAGE protocols can be

found on page 18.

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Handy Hint: The Phos-tag requires two dianions, so the molar ratio of MnCl2 is twice that

of the Phos-tag. The protocol can also be adapted to use Zn2+ - see page 13.

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Handy Hint: For high molecular weight proteins, 200-350 kDa, low concentration

acrylamide gels can be strengthed with agarose – see adapted method on page 13.

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Quick Guide for Resolving Gels (10mL)

Phos-tag™ Acrylamide conc. 20µM 50µM 100µM

Acrylamide conc. 12% 10% 8% 6% 12% 10% 8% 6% 12% 10% 8% 6%30% Acrylamide solution (mL) 4 3.33 2.67 2 4 3.33 2.67 2 4 3.33 2.67 2

1.5M Tris HCl pH 8.8 (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

5mM Phos-tag Acrylamide (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

10mM MnCl2 (mL) 01 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

10% SDS (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

10% ammonium persulphate 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

Sample Preparation & electrophoresis

1. Mix sample 2:1 with loading buffer in a microcentrifuge tube and heat at 95°C for 5 min; e.g. 6 µL sample and 3.0 µL buffer.

2. Allow the solution to cool to room temperature.

3. Assemble the electrophoresis equipment and fill the electrode chambers with running buffer.

4. Gently remove the comb from the stacking gel and load the samples into the wells using a micropipette.

5. Attach the leads to power supply and run the gels under a constant current condition (30 mA/gel) until the Bromophenol blue dye front reaches the bottom of the resolving gel.

Gel Staining

1. Once electrophoresis is completed soak the gel in 50ml acidic protein fixation solution for 10 min with gentle agitation.

2. Stain the gel by soaking in 50ml Coomassie Brilliant Blue Stain for 2 h with gentle agitation.

3. Wash the gel in 50ml Destaining Solution x 3 to remove excess stain and until the background is sufficiently clear.

Western Blotting

1. When electrophoresis is complete soak the gel in 50ml transfer buffer with 1-10mM EDTA for 10 min x 3 to eliminate the manganese ions prior to transfer. This improves the transfer efficiency. (Running buffer can be used in place of transfer buffer if preferred).

2. Wash the gel in 50ml transfer/running buffer for 10 min to remove EDTA before blotting onto a PVDF membrane.

Handy Hint: EDTA, inorganic salts and surfactant within the sample can lead to

distortion of the protein bands during electrophoresis.

Desalting of samples is recommended.

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Handy Hint: Silver stain or fluorescent stains can also be used.

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Handy Hint: Wet tank transfer is the preferred method but semi-dry blotting can also

be used.

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Handy Hint: Zn2+ Phos-tag SDS-PAGE gels are more stable and do not deteriorate

as quickly as Mn2+ gels so can be cast in advance.

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Preparing Zn2+ Phos-tag SDS-PAGE Gels

1. The procedure for casting gels is the same as with Mn2+ Phos-tag SDS-PAGE but uses the following gel solutions:

Resolving Gel Solution (10 mL: 12% (w/v) Acrylamide and 50 µmol/L Phos-tagTM AAL)

30% (w/v) Acrylamide Solution 4.00 mL

1.4 mol/L Bis-Tris/HCl Solution, pH 6.8 2.50 mL

5.0 mmol/L Phos-tag AAL Solution 0.10 mL

10 mmol/L ZnCl2 Solution 0.10 mL

TEMED (tetramethylethylenediamine) 10 µL

Distilled Water 3.24 mL

Mix reagents and degas for 2 min whilst stirring. Add 20-50µl of 10% Ammonium Persulphate Solution and stir gently to mix. Pour and top with a layer of butanol-saturated water. Leave to polymerise for 1 h at room temperature.

2. Wash with distilled water to remove butanol layer. Blot dry and top with stacking gel.

Stacking Gel Solution (10mL: 4.5% (w/v) Acrylamide)


1.4 mol/L Bis-Tris/HCl Solution, pH 6.8 2.50 mL



Mix reagents and degas for 2 min whilst stirring. Add 20-50µl of 10% Ammonium Persulphate Solution and stir gently to mix. Pour and leave to polymerise for 1 h at room temperature.

Preparing Mn2+ Phos-tag SDS-PAGE Gels for High Molecular Weight Samples

1. To prepare Mn2+ Phos-tag SDS-PAGE for high molecular weight sample the low percentage acrylamide gels can be strengthened with agarose. To prepare the resolving gel:

Resolving Gel Solution (10 mL: 3% (w/v) Acrylamide, 0.5%(w/v) agarose and 20 µmol/L Phos-tag AAL)


1.5 mol/L Tris/HCl Solution, pH 8.8 2.50 mL

5.0 mmol/L Phos-tag AAL Solution 0.04 mL

10 mmol/L MnCl2 Solution 0.04 mL

10% (w/v) SDS Solution 0.10 mL



Mix the acrylamide solution and put to one side whilst then prepare the agarose solution. Dissolve 0.3g agarose in 20ml of distilled water to give a 1.5% (w/v) solution. Add 3.3ml of the hot agarose solution and mix carefully to avoid bubbles. Add 50µL of 10% Ammonium Persulphate Solution, stir gently and pour.

Once the gel has cooled a 2-3mm layer of agarose containing unpolymerised acrylamide will remain at the top of the gel, add 100µL distilled water and scrape the gel. Blot dry with tissue. This needs to be removed in order for the stacking gel to adhere to the resolving gel.

Handy Hint: Remember to use the Tris-MOPS running buffer with Zn2+ Phos-tag

SDS-PAGE gels.

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Handy Hint: Do not overlay the resolving gel with liquid as this will give them a ragged

edge, leave to air dry.

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Handy Hint: Once cool the acrylamide/agarose gels can be stored over night. Add a layer

of distilled water to the gel to stop it dying out.

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2. Prepare stacking gel and pour on top of resolving gel.

Stacking Gel Solution (10ml: 3% (w/v) Acrylamide, 0.5%(w/v) agarose)


0.50 mol/L Tris/HCl Solution, pH 6.8 2.50 mL

10% (w/v) SDS Solution 0.10 mL



Mix the above reagents, then add 3.33ml hot 1.5% (w/v) agarose. Mix before adding 50µl of 10% Ammonium Persulfate Solution. Stir gently and pour on top of resolving gel and add a comb. Leave to set for 20 min.

Western Blotting with Phos-tag Biotin ProtocolE. Kinoshita-Kikuta, E. Kinoshita & T. Koike (Hiroshima University) Ver 8 (2013/4)

http://home.hiroshima-u.ac.jp/tkoike/pro/PT_CL_e.pdf

Preparing Phos-tag Biotin Streptavadin-conjugated HRP Complex

1. Prepare a 4:1 complex of Phos-tag-Biotin and Streptavidin-conjugated HRP:

Tris Buffered Saline-0.01% Tween 20 (TBS-T) 469 µL

Phos-tag Biotin solution (BTL-104/105/111) 1 ~ 10 µL

10 mmol/L Zn(NO3)2 20 µL

Streptavidin-conjugated Horseradish Peroxidase 1 µL

Mix and leave to equilibrate for 30 min at room temperature.

2. The Phos-tag Biotin and Streptavadin – conjugated HRP is then subjected to centrifugal filtration to remove excess Phos-tag Biotin. For example using (NMWL = 30,000, NanosepTM 30K, Pall Life Sciences). Centrifuge (14,000×g) for 20 min at room temperature.

3. The remaining solution (<10 µL) is diluted with 30 mL of 1xTBS-T. (Phos-tag BTL)4–(Streptavidin-conjugated HRP) is stable for 30 days at 4 °C.

Probing Western Blots with Phos-tag-bound Streptavidin-conjugated HRP

1. Soak the protein-blotted PVDF membrane with 1xTBS-T for 1 h with gentle rocking. The membrane is gently rocked for at least 1 h.

2. The membrane is then incubated with Phos-tag Biotin Streptavidin-conjugated HRP, allow 1ml/5cm2 membrane and seal in a plastic bag. Leave for 30 min with gentle rocking.

3. The membrane is taken out of the bag and washed twice with TBS-T (allow 10 mL/5 cm2) for 5 min.

4. Then to detect the target proteins, the blot is incubated with chemiluminescence reagents and wrapped in film. X-ray film is exposed to the blot and then developed to visualise the chemiluminescence.

Handy Hint: Re-melt the unused agarose you prepared for the the resolving gel.

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Handy Hint: Phos-tag Biotin BTL-104 and BTL-105 need to be dissolved in methanol and

diluted with TBS before use. BTL-111 is supplied as a 1mM solution.

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Handy Hint: Recipes for solutions used in Phos-tag Biotin Western blotting protocol can

be found on page 20.

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Phosphate Affinity Chromatography Protocols E. Kinoshita-Kikuta, E. Kinoshita & T. Koike (Hiroshima University) Ver 6 (2010/5)

http://home.hiroshima-u.ac.jp/tkoike/pro/PT_PAC_e.pdf

Preparation of Samples for Phosphate Affinity Chromatography

Samples must be prepared with a suitable lysis buffer, such as RIPA buffer, to avoid excess amounts of Zn2+chelating agents (e.g. 5mM EDTA) or phosphate derivatives in the sample. See below for guidance on sample preparation reagents.

Using a 100mm culture plate of cultured cells, such as A431 human epidermoid carcinoma cells as an example, samples can be prepared as follows:

1. Remove culture media and rinse the cells with 20 mmol/L Tris-HCl (pH 7.6) buffer containing 138 mmol/L NaCl. Do not use a phosphate-containing buffer.

2. Incubate cells in 0.30ml of cold RIPA buffer and gently rock for 15 min on ice.

3. Remove the cells using a cell scraper and transfer to a centrifugation tube.

4. Wash the plate with 0.20ml cold RIPA buffer and add to the tube.

5. Incubate the cell suspension on ice for 60 min.

6. Centrifuge the tube at 10000×g for 10 min at 4°C.

7. The resulting supernatant fluid should contain approximately 1 mg of solubilised proteins.

8. Use a protein quantification assay, such as the Bradford method, to determine the protein concentration of the supernatant and adjust to give approximately 2mg/ml.

9. Adjust the concentration of the solubilised proteins to ~ 2 mg/mL with an appropriate amount of an RIPA buffer.

10. Dilute 0.2ml of the resulting solution 0.8mL of Binding/Washing buffer to obtain the sample for column chromatography.

Category Reagent Allowable concentrationsReducing agents DTT ≤ 0.1 M

Denaturing agents Urea Using it at 8 M has no negative effect

Surfactants (anionic)SDS Using it at ≥ 0.5% affects the binding process

Sodium deoxycholate Using it at ≥ 0.25% affects the binding process

Surfactants (nonionic)Nonidet P40 ≤ 1%

Tween 20 ≤ 1%

Surfactants (amphoteric) CHAPS ≤ 0.2%

Phosphate derivativesß-Glycerophosphate Do not use it

Pyrophosphate Do not use it

Chelating agents EDTA Using it at a high concentration has a negative effect

Handy Hint: Recipes for solutions used in Phos-tag Agarose chromatography

protocols can be found on page 21.

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Preparation of a Zn2+–Phos-tag Agarose Chromatography Column

1. Remove the cap of a polypropylene 1-mL column for gravity-flow chromatography.

2. Wash the column with approximately 1ml 2-propanol.

3. Dip the column in distilled water to remove air from the column filter.

4. Place Phos-tag agarose in the column to make 1.0 mL of a compressed gel bed.

5. Set the column on a column stand and wash with Phosphoprotein Binding/Washing buffer (1 mL ×3). The flow rate is 0.1 ~ 0.5 mL/min.

6. Apply Phosphoprotein Balancing buffer (1 mL ×2).

7. Wash the gel bed with Phosphoprotein Binding/Washing buffer (2 mL ×3). The column is now ready to use.

Separation of Phosphoproteins from Cell Lysate

1. Apply a cell lysate sample, 1 mL, containing 0.3 ~ 0.5 mg proteins.

2. Collect a flow-through fraction (1 mL).

3. Wash the protein-bound column with Phosphoprotein Binding/Washing buffer (1 mL ×5). Collect five washing fractions. (Check the content by an appropriate method).

4. Elute phosphorylated proteins with Phosphoprotein Elution buffer (1 mL ×5). Collect five elution fractions. (Check content by an appropriate method).

5. The obtained phosphorylated proteins in the elution fractions can be desalted and condensed using an ultrafiltration unit for downstream Western blot analysis.

Enrichment of Phosphorylated Peptides using a Spin Column

1. Wash a spin column unit with 2-propanol. Place 60 µL of 50%(v/v) Phos-tag Agarose into the sample reservoir of the spin column unit and centrifuge the spin column unit for 20 s.

2. Discard the filtrate. Apply 0.10 mL of the Phosphorylated Peptide balancing buffer in the sample reservoir. After 5 min centrifuge the unit for 20 s and discard the filtrate.

3. Wash the gel twice with 0.10 mL of the Phosphorylated Peptide binding/washing buffer by the same centrifugal method.

4. Apply a solution (0.10 mL) of protein tryptic digest, which contains less than 20 µg peptides. After equilibration for 5 min centrifuge the spin column and collect a flow-through fraction as the filtrate.

5. Wash the phosphopeptide-bound gel three times with 0.10 mL of the Phosphorylated Peptide binding/washing buffer and obtain three washing fractions.

6. Elute phosphorylated peptides four times with 0.10 mL of elution buffer and collect four elution fractions. The obtained phosphorylated peptides can be desalted and condensed using a hydrohobic resin for downstream analysis.

Handy Hint: The volume of Phos-tag Agarose in this protocol is 1ml, but it can be

optimised in proportion to the amount of sample.

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Handy Hint: After using the column discard. Do not re-use as the column efficiency of the

used gel is much lower due to residual compounds in the gel.

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Handy Hint: When a larger amount of protein is applied leakage of phosphoproteins in

the flow-through and washing fractions may occur.

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Handy Hint: Some proteins adsorb on the centrifugal filter unit. Estimate the recovery

of eluted proteins using a protein quantification assay.

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Handy Hint: A number of elution buffers can be used:

0.20M Na2HPO4-NaOH (pH 7)

5%(w/v) aqueous NH3 (1.0 mL of 28%(w/w) aqueous NH3 + 4.0 mL of distilled water)

1%(v/v) H3PO4 (50 µL of H3PO4 + 4.95 mL of distilled water)

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Handy Hint: Addition of 10-50%(v/v) CH3CN would increase recovery.

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Suggested matricesMALDI-TOF Mass Analysis of Phosphorylated Molecule ProtocolE. Kinoshita-Kikuta, E. Kinoshita & T. Koike (Hiroshima University) Ver 2 (2010/6)

http://home.hiroshima-u.ac.jp/tkoike/pro/PT_MS_e.pdf

Sample Solution

Because the Phos-tag molecule selectively captures target phosphate in a pH range of 5 - 8, the buffer solution should be employed for the 1:1 complexation. If you are examining a sample mixture, it may be necessary to prepare the mixture with several different matrices in an appropriate buffer solution.

The following buffer systems are recommended:

10 mM Tris-borate (pH 8) or

Good’s buffers such as: 10 mM HEPES/NaOH (pH 7.5)

10 mM TAPS/NaOH (pH 8.4)

10 mM MES/NaOH (pH 6.2)

Note: Do not use inorganic phosphate for the buffer system, which is a competitive ligand to the Phos-tag molecule.

A high salt concentration may interfere with sample ionisation and increase the adduct peaks such as Na+-M and K+-M.

Matrix Solution

The matrix molecule for MALDI-TOF MS plays a key role not only in the ionisation process, but also in the complexation of a target phosphate with Phos-tag. The phenolic or amine matrices (see opposite) are recommended for phosphorylated peptides and phospholipids (e.g. lysophosphatidic acid). The matrix solution (10 ~ 40 mg/mL) in an appropriate organic solvent should be prepared prior to use.

Note: Do not use acidic matrices such as cinnamic acid derivatives.

Phos-tag Solution

The stock solution of 1.0 mM Phos-tag MS-101 (acetate-bound form) is prepared using deionised water. Do not adjust pH with acid or base. The solution pH is around 6. Store at 4°C.

Sample Preparation on the Sample Plate

Mix sample solution, matrix solution and Phos-tag solution directly on the sample plate (the total volume approximately 1 µL). The volume ratio of the solutions is optimised for each sample in a similar manner as reported for the general MALDI-TOF MS analysis. The solvent of the mixture is evaporated under an appropriate condition. Load the sample plate in the mass spectrometer.

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Solutions required for Mn2+ Phos-tag SDS-PAGE30% (w/v) Acrylamide Solution (30% T, 3.3% C) Acrylamide 29.0 g N,N’-methylene-bisacrylamide 1.0 g Make to 100 mL with distilled water. Filter and store at 4°C in the dark.

1.5 mol/L Tris/HCl Solution, pH 8.8 (4x solution for resolving gel) Tris base (MW: 121, pKa = 8.2 at 20°C) 18.2 g 6.0 mol/L HCl (0.19 equivalents of Tris) 4.85 mL Make to 100 mL with distilled water. Store at 4°C. 0.50 mol/L Tris/HCl Solution, pH 6.8 (4x 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 Carefully adjust to pH 6.8 (non-buffered pH region) with 6.0 mol/L HCl (ca. 0.1 mL). Bring to total volume to 100 mL with distilled water. Store at 4°C. 10% (w/v) SDS Solution Dissolve 10.0 g SDS in 90 mL of distilled water with stirring and bring to total volume to 100 mL with distilled water. Store at 4°C. 5.0 mmol/L Phos-tag AAL Solution containing 3% (v/v) MeOH Phos-tag AAL-107 10 mg* Methanol 0.10 mLDistilled water 3.2 mL Dissolve the oily Phos-tag AAL-107 product (10 mg) in 0.10 mL methanol. The solution is then diluted with 3.2 mL distilled water by pipetting.

*[NB please adjust volumes accordingly if using the 2mg vial] 10 mmol/L MnCl2 Solution Dissolve 0.10 g MnCl2(H2O)4 (MW: 198) in 50 mL of distilled water.

Note: Do not use another anion salt, such as Mn(NO3)2 and Mn(CH3COO)2.

10% (w/v) Ammonium Persulfate Solution Dissolve 10 mg (NH4)2S2O8 (MW: 228) in 0.10 mL of distilled water.

Note: Freshly prepare prior to use. Running Buffer, pH 8.3 (10x solution) Tris base (0.25 mol/L) 15.1 g SDS 5.0 g Glycine (1.92 mol/L) 72.0 g Make to 0.50 L with distilled water. Do not adjust pH with acid or base. Store at 4°C. Dilute 1:9 before use.

Sample Buffer (3x solution) Bromophenol Blue (BPB, a tracking dye) 1.5 mg SDS 0.60 g Glycerol 3.0 mL 0.50 mol/L Tris/HCl, pH 6.8 3.9 mL 2-mercaptoethanol 1.5 mL

Make to 10 mL with distilled water. Store at –20°C.

Solutions

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Acidic Solution for Fixation of Proteins (1 L) Acetic acid 0.10 L Methanol 0.40 L Distilled water 0.50 L 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

After dissolving CBB in methanol, acetic acid and water are added into the solution. Washing and Destaining Solution (1 L) Methanol 0.25 L Acetic acid 0.10 L Distilled water 0.65 L

Solutions required for Zn2+ Phos-tag SDS-PAGE10 mmol/L ZnCl2 Solution Dissolve 0.70 g ZnCl2 (MW: 136, purity >98%) in 500 mL of distilled water.

Note: Because zinc(II) chloride is a deliquescent salt, the ZnCl2 solution should be prepared using a fresh product in a new bottle. If an insoluble material such as a small amount of ZnO (impurity) remains in the solution, it should be removed by filtration.

1.4 mol/L Bis-Tris/HCl Solution, pH 6.8 (4x solution) Bis-Tris base (MW: 209, pKa = 6.5 at 20°C) 29.9 g 6.0 mol/L HCl (0.42 equivalent of Bis-Tris) 10 mL

Make up to 100 mL with distilled water. Store at 4°C.

Note: This buffer solution is used for the resolving and stacking gels.

0.5 mol/L sodium bisulfite solution NaHSO3 (MW: 106) 5.3 g

Make to 100 mL with distilled water. Store at 4°C.

Sulfite ion (SO32-) is a reducing reagent diminishing O2 in the electrode buffer and inhibits the oxidation of reduced proteins in the

gel. The stock solution should be stored in a glass bottle tightly capped.

Running Buffer, pH 7.8 (5x solution) Tris base (MW: 121, pKa = 8.2 at 20°C, 0.50 mol/L) 30.3 g MOPS (MW: 209, pKa = 7.2 at 20°C, 0.50 mol/L) 52.3 g 10% (w/v) SDS Solution (0.5% (w/v)) 25.0 mL

Make to 0.50 L with distilled water. Do not adjust pH with acid or base. Store at 4°C. Dilute 100 mL of running buffer with 5 mL of 0.5 mol/L NaHSO3 and 395 mL distilled water prior to use (Total volume: 0.50 L).

Solutions

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Solutions

Solutions required for Western Blotting with Phos-tag BiotinTris buffered saline (10xTBS, 1 L, pH 7.5) Tris (0.10 mol/L) 12.1 g NaCl (1.0 mol/L) 58.4 g Distilled water 0.9 L 2 mol/L aqueous HCl for pH adjustment

Once pH is adjusted to 7.5 make up to 1L with distilled water.

Dilute 1:9 for 1xTBS. Add 1ml 10% (v/v) Tween 20 solution to 1L of TBS to give TBS-T solution.

10% (v/v) Tween 20 solution (50 mL) Tween 20 5 mL Distilled water 45 mL Phos-tag BTL Methanol SolutionPhos-tag BTL-104 BTL-105 10 mg Methanol 0.13 mL

Phos-tag BTL solution 10mmol/LPhos-tag BTL-104 0.13 mL1x TBS 1.17 mL

Or

Phos-tag BTL-105 0.13 mL1x TBS 1.00 mL[Phos-tag Biotin BTL-111 is supplied as 1mmol/L solution]

10 mmol/L Zn(NO3)2 aqueous solution (50 mL)

Zn(NO3)2(H2O)6 (MW. 297) 0.15 g Distilled water 50 mL (An alternative is an aqueous solution of 10 mmol/L ZnCl2.)

Stripping buffer (1 L) 0.50 mol/L Tris/HCl Solution, pH 6.8 125 mL 10% (w/v) SDS Solution 200 mL 2-mercaptoethanol 7 mL Distilled water 668 mL

Running Buffer, pH 7.8 (5x solution) Tris base (MW: 121, pKa = 8.2 at 20°C, 0.50 mol/L) 30.3 g MOPS (MW: 209, pKa = 7.2 at 20°C, 0.50 mol/L) 52.3 g 10% (w/v) SDS Solution (0.5% (w/v)) 25.0 mL

Make to 0.50 L with distilled water. Do not adjust pH with acid or base. Store at 4°C. Dilute 100 mL of running buffer with 5 mL of 0.5 mol/L NaHSO3 and 395 mL distilled water prior to use (total volume: 0.50 L).

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Solutions

Phoshate affinity chromotography with Phos-tag agarose1.0 M Tris-CH3COOH buffer (pH 7.5, 100 mL) Tris: tris(hydroxymethyl)aminomethane 12.1 g Distilled water 70 mL 3.0 mol/L aqueous CH3COOH for pH adjustment

Add distilled water to bring to 100 mL. Phosphoprotein Binding/Washing buffer (0.10 mol/L Tris-CH3COOH, 1.0 mol/L CH3COONa, pH 7.5, 100 mL) 1.0 M Tris-CH3COOH buffer (pH 7.5) 10 mL CH3COONa•3H2O (MW: 136) 13.6 g Distilled water 80 mL 3.0 mol/L aqueous CH3COOH for pH adjustment

Add distilled water to bring to 100 mL. Balancing buffer (20 µmol/L Zn(CH3COO)2, pH 7.5, 5 mL) Binding/Washing buffer 5 mL 10 mmol/L aqueous Zn(CH3COO)2 10 µL

0.20 M NaH2PO4-NaOH buffer (pH 7.5, 100 mL) NaH2PO4 2.4 g Distilled water 80 mL 1.0 mol/L aqueous NaOH for pH adjustment

Add distilled water to bring to 100 mL.

Phosphoprotein Elution buffer (0.10 mol/L Tris-CH3COOH , 1.0 mol/L NaCl, 10 mmol/L NaH2PO4 -NaOH, pH 7.5, 50 mL) 1.0 M Tris-CH3COOH buffer (pH 7.5) 5.0 mL NaCl 2.9 g Distilled water 38 mL 0.20 M NaH2PO4-NaOH buffer (pH 7.5) 2.5 mL 3 mol/L aqueous CH3COOH for pH adjustment

Add distilled water to bring to 50 mL. RIPA buffer (a radio-immunoprecipitation assay lysis buffer) 50 mmol/L Tris-HCl (pH 7.4) containing 0.15 mol/L NaCl, 0.25%(w/v) sodium deoxycholate, 1.0%(v/v) Nonidet P-40, 1.0 mmol/L EDTA, 1.0 mmol/L phenylmethanesulfonyl fluoride, 1 µg/mL Aprotinin, 1 µg/mL Leupeptin, 1 µg/mL Pepstatin, 1.0 mmol/L Na3VO4 and 1.0 mmol/L NaF

Do not use excess amount of a zinc(II)-chelating agent (e.g., 5 mM EDTA) and/or phosphate derivative (e.g. inorganic phosphate and nucleotides) in the sample.

10 mmol/L MES-NaOH, 0.10 mol/L NaCl, 5.0 mmol/L Na2C2O4 (pH 6.0, 100 mL) MES: 2-(N-morpholino)ethanesulfonic acid (MW: 195) 2.0 g NaCl (MW: 58.4) 0.58 g Na2C2O4 (sodium oxalate, MW: 134) 67 mg Distilled water 100 mL 0.10 mol/L aqueous NaOH for pH adjustment

Add distilled water to bring to 100 mL.


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Phosphorylated Peptide Binding/Washing buffer (10 mL) 10 M MES-NaOH, 0.10 M NaCl, 5.0 M Na2C2O4 (pH 6.0) 10 ~ 5 mL 0 ~ 50%(v/v) CH3CN * 0 ~ 5 mL

*The optimum ratio of CH3CN should be determined for each phosphorylated peptide.

Balancing buffer (5 mL) Binding/Washing buffer (10 mL) 5 mL 10 mmol/L aqueous Zn(CH3COO)2 10 µL

Phosphorylated Peptide Elution buffers (5 mL): No.1. 0.20 mol/L Na2HPO4-NaOH (pH 7)

Or

No.2. 5%(w/v) aqueous NH3 (1.0 mL of 28%(w/w) aqueous NH3 + 4.0 mL of distilled water)

Or

No.3. 1%(v/v) H3PO4 (50 µL of H3PO4 + 4.95 mL of distilled water)

Addition of 10 ~ 50%(v/v) CH3CN would increase the recovery of phosphorylated peptides. No.2 is well suited for the MALDI-TOF mass analysis.

Solutions

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Bibliography

Phos-tag™ BibliographyPhos-tag was discovered in Japan and as such many of the papers included in this selected bibliography originate from Japanese research establishments. However, researchers throughout the World have since been discovering the power of Phos-tag technology for themselves, including some UK groups, which have been highlighted.

Phos-tag Acrylamide

Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells. Ghalayini, M.K. , Dong, Q., Richardson, D.R., & Assinder, S.J. Biosci Rep (2013): 33(3) art:e00042.doi:10.1042/BSR20130042

Mechanism of activation of PhoQ/PhoP two-component signal transduction by SafA, an auxiliary protein of PhoQ histidine kinase in Escherichia coli. Ishii, E., Eguchi, Y., & Utsumi, R. Biocsi Biotechnol Biochem (2013): 77(4): 814-9

Phosphorylation-dependent localization of the response regulator FrzZ signals cell reversals in Myxococcus xanthus. Kaimer, C. & Zusman, D.R. Mol Microbiol (2013): 88(4): 740-53

Different signalling mechanisms are involved in the norepinephrine-stimulate TORC1 and TORC2 nuclear translocation in rat pinealocytes. McTague, J. Amyotte, N., Kanyo, R., Ferguson, M., Chik, C.L., & Ho, A.K.. Endocrinology (2012):153(8): 3839-49

Protein kinase A is central for forward transport of two-pore domain potassium channels K2P3.1 and K2P9.1. Mant, A., Elliott, D., Eyers, P.A., & O’Kelly, I.M. J Biol Chem (2011):286(16):14110-9

Quantitative measurement of in vivo phosphorylation states of Cdk5 activator p35 by Phos-tag SDS-PAGE. Hosokawa, T., Saito, T., Asada, A., Fukunaga, K., & Hinsanaga, S. Mol Cell Proteomics (2010): 9: 1133-1143

Sustained Mps1 activity is required in mitosis to recruit O-Mad2 to the Mad1-C-Mad2 Core complex. Hewitt, L., Tighe, A., Santaguida, S. White, A.M., Jones, C.D, Musacchio, A., Green, S., & Taylor, S.S. J Cell Biol (2010): 190(1):25-34

Characterization of multiple alternative forms of heterogeneous nuclear ribonucleoprotein K by phosphate-affinity electrophoresis. Kimura, Y., Nagata, K., Suzuki, N., Yokoyama, R., Yamanaka, Y., Kitamura, H., Hirano, H., & Ohara, O. Proteomics (2010): 10(21): 3884-95

Inhibition of GSK-3 ameliorates Abeta pathology in an adult-onset Drosophila model of Alzheimer’s disease. Sofola, O., Kerr, F., Rogers, I., Killick,R., Augustin, H., Gandy, C., Allen, M.J., Hardy, J., Lovestonem S., & Partridge, L. PLoS Genet (2010):6(9):e1001087

Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE. Kinoshita, E., Kinoshita-Kikuta, E., & Koike, T. Nature Protoc (2009): 4(10):1513-21

The use of phosphate-affinity SDS-PAGE to measure the cardiac troponin I phosphorylation site distribution in human heart muscle. Messer, A.E., Gallon, C.E., McKenna, W.J., Dos Remedios, C.G., Marston, S.B. Proteomics Clin Appl (2009): 3(12) :1371-82

A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel electrophoreseis. Kinoshita-Kikuta, E., Kinoshita, E., & Koike, T. Anal Biochem (2008): 378(1):102-4

A single nucleotide polymorphism genotyping method using phosphate-affinity polyacrylamide gel electrophoresis. Kinoshita, E., Kinoshita-Kikuta, E., and Koike, T. Anal Biochem (2007):361(2):294-8

Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K., & Koike, T. Mol Cell Proteomics (2006): 5(4): 749-57.

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Phos-tag Agarose

Integrated quantitative analysis of the phosphoproteome and transcriptome in tamoxifen-resistant breast cancer. Oyama, M., Nagashima, T., Suzuki, T., Kozuka-Hata, H., Yumoto, N., Shiraishi, Y., Ikeda, K., Kuroki, Y., Gotoh, N., Ishida, T., Inoue, S., Kitano, H., & Okada-Hatakeyama, M. J Biol Chem (2011):286(1):818-829

Identification on membrane and characterization of phosphoproteins using an alkoxide-bridged dinuclear metal complex as a phosphate-binding tag molecule. Nakanishi, T., Ando, E., Furuta, M., Kinoshita, E., Kinoshita-Kikuta, E. Koike, T., Tsunasawa, S., & Nishimura, O. J Biomol Tech (2007): 18(5): 278-86

Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins. Kinoshita, E., Yamada, A., Takeda, H., Kinoshita-Kikuta, E., & Koike, .T. J Sep Sci (2005):28(2): 155-62

Phos-tag Mass Analytical Kit

Identification of a sphingolipid-specific phospholipase D activity associated with the generation of phytoceramide-1-phosphate in cabbage leaves. Tanaka, T., Imai, H., Morishige, J.I., Yamashita, R., Matsuoka, H., Uozumi, S., Satouchi, K., Nagano, M. & Tokumura, A. FEBS J (2013) : doi: 10.1111/febs.12374

A clean-up technology for the simultaneous determination of lysophosphatidic acid and sphingosine-1-phosphate by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a phosphate-capture molecule, Phos-tag. Morishige, J., Urikura, M., Takagi, H., Hirano, K., Koike, T., Tanaka, T., & Satouchi, K. Rapid Commun Mass Spectrom (2010): 24(7): 1075-84

Microfabricated dual sprayer for on-line mass tagging of phosphopeptides. Prudent, M., Rossier, J.S., Lion, N. & Girault, H.H. Anal Chem (2008): 80(7):2531-8

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of phosphorylated compounds using a novel phosphate capture molecule. Takeda, H., Kawasaki, A., Takahashi, M., Yamada, A., & Koike, T. Rapid Commun Mass Spectrom (2003): 17(18):2075-81

Phos-tag Biotin

Sandwich assay for phosphorylation of protein multiplexes by using antibodies and Phos-tag. Kinoshita, E., Kinoshita-Kikuta, E., & Koike, T. Anal Biochem (2013): 438(2): 104-6

Highly sensitive detection of protein phosphorylation by using improved Phos-tag Biotin. Kinoshita, E., Kinoshita-Kikuta, E., Sugiyama, Y. Fukada, Y., Ozeki, T., & Koike, T. Proteomics (2012): 12(7): 932-7

Protein kinase substrate profiling with a high-density peptide microarray. Comb Chem High Throughput Screen. Han,X., sonoda, T., Mori, T., Yamanouchi, G., Yamaji, T., Shigaki, S., Niidome, T. & Katayama, Y. (2010): 13(9):777-89.


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