phos-tag™ sds-page guidebook - wako-chem.co.jp .compound designed from an alkaline phosphatase
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TM
Phos-tag SDS-PAGE GUIDEBOOK
phos-tag.jpphos-tag.jp
16901GF
INDEX
1Principle and Application 2Phos-tag SDS-PAGE is 3Protocol Mn2+-Phos-tag SDS-PAGE
Zn2+-Phos-tag SDS-PAGE
4Trouble Shooting 5Optimization of Phos-tag SDS-PAGE Condition 6Application Data and References 7FAQ (Phos-tag Series) 8SuperSep Phos-tag 9. Phos-tag Series 10. Related ProductsElectrophoresis/Western Blotting)
1Principle and A
pplication
P3
P4
P6
P6
P11
P14
P17
P18
P22
P26
P27
P29Phos-tag was developed by the Department of Functional Molecular Science atHiroshima University. http://www.phos-tag.com/
Phos-tag is
Basic Structure of Phos-tag
Phos-tag is a functional molecule which captures phosphorylated Ser/Thr/Tyr and His/Asp/Lys. It is a synthesized chemical compound designed from an alkaline phosphatase catalytic domain as a model. A series of reagents utilizing Phos-tag can be used in the separation, detection, mass spectrometry analysis, and purification of phosphorylated proteins.
Product Purpose of UseSeparation Separation is possible by SDS-PAGE
depending on the degree of phos-phorylation.
Purification Phosphorylated proteins are purified by column chromatography.
Analysis This is used in MALDI-TOF/MS analysis to improve the detection sensitivity of phosphorylated molecules.
Ready-to-use precast gel containing 50 M Phos-tag Acrylamide
Separation
Phos-tag Acrylamide
Phos-tag Agarose
Purification Ready-to-use tip to purify phosphory-lated peptides
Phos-tag Tip
Phos-tag Mass Analytical Kit
SuperSep Phos-tag
Phos-tag BiotinDetection A substitute for the anti-phospho
antibody used in western blot.
TM
Principle
Two metallic ions cooperateto bind a phosphate group
Application Time-course of -casein dephosphorylation
1Principle and Application
M2+Zinc ion or manganese ion Selectivity of binding of a phosphate ion
(2-) is much higher than that of other
anions.
Stable complex is formed under physi-
ological conditions (pH 5 to 8).
Non-phosphorylated protein
Phosphorylated protein
1. Divalent metal ions trap phosphorylated proteins during migration.2. The higher the amount of phosphorylation, the slower the migration velocity.
3. Separation occurs based on phosphorylation levels.(Separation can occur if the phosphorylation sites are different, even with identical levels of phosphorylation)
MTM
1. D2. Thve
3. Se(Sev
Phosphorylated forms can be separated by the amount and
site of phosphorylation.
12001200Phosphatase-treated time
Phosphorylated -casein
Non-phosphorylated -casein
Phosphorylated -casein+
Non-phosphorylated -casein
Conventional SDS-PAGE Phos-tag SDS-PAGE 10 % Acrylamide 10 % Acrylamide
100 M Mn2+-Phos-tagTM Acrylamide
Phos-tag SDS-PAGE and conventional SDS-PAGE was used to separate -casein samples, which have been dephosphorylated over time (incubation duration: 0120 min) by alkaline phosphatase.
Phos-tag ApplicationsMultiple possibilities of Phos-tag
Expressed Proteins Cells Animal tissues
Identification of new phosphoproteins
10. Related ProductsElectrophoresis/Western Blotting) P29
Phos-tag ApplicationsMultiple possibilities of Phos-tag
Expressed Proteins Cells Animal tissues
Identification of new phosphoproteins
Ready for research of phosphorylation at various stages and for various purposes
No need for applying radioactivity
Kinase Assay
Analysis of Kinase catalyzed signaling cascades.
Analysis of phosphorylation status of endogenous proteins
Increase / Decrease of phosphorylation by stimulation
Analysis of genetically modified mice
No need for preparation of Anti phospho-Antibodies
phos-tag.jp phos-tag.jp
INDEX
1Principle and Application 2Phos-tag SDS-PAGE is 3Protocol Mn2+-Phos-tag SDS-PAGE
Zn2+-Phos-tag SDS-PAGE
4Trouble Shooting 5Optimization of Phos-tag SDS-PAGE Condition 6Application Data and References 7FAQ (Phos-tag Series) 8SuperSep Phos-tag 9. Phos-tag Series 10. Related ProductsElectrophoresis/Western Blotting)
1Principle and A
pplication
P3
P4
P6
P6
P11
P14
P17
P18
P22
P26
P27
P29Phos-tag was developed by the Department of Functional Molecular Science atHiroshima University. http://www.phos-tag.com/
Phos-tag is
Basic Structure of Phos-tag
Phos-tag is a functional molecule which captures phosphorylated Ser/Thr/Tyr and His/Asp/Lys. It is a synthesized chemical compound designed from an alkaline phosphatase catalytic domain as a model. A series of reagents utilizing Phos-tag can be used in the separation, detection, mass spectrometry analysis, and purification of phosphorylated proteins.
Product Purpose of UseSeparation Separation is possible by SDS-PAGE
depending on the degree of phos-phorylation.
Purification Phosphorylated proteins are purified by column chromatography.
Analysis This is used in MALDI-TOF/MS analysis to improve the detection sensitivity of phosphorylated molecules.
Ready-to-use precast gel containing 50 M Phos-tag Acrylamide
Separation
Phos-tag Acrylamide
Phos-tag Agarose
Purification Ready-to-use tip to purify phosphory-lated peptides
Phos-tag Tip
Phos-tag Mass Analytical Kit
SuperSep Phos-tag
Phos-tag BiotinDetection A substitute for the anti-phospho
antibody used in western blot.
TM
Principle
Two metallic ions cooperateto bind a phosphate group
Application Time-course of -casein dephosphorylation
1Principle and Application
M2+Zinc ion or manganese ion Selectivity of binding of a phosphate ion
(2-) is much higher than that of other
anions.
Stable complex is formed under physi-
ological conditions (pH 5 to 8).
Non-phosphorylated protein
Phosphorylated protein
1. Divalent metal ions trap phosphorylated proteins during migration.2. The higher the amount of phosphorylation, the slower the migration velocity.
3. Separation occurs based on phosphorylation levels.(Separation can occur if the phosphorylation sites are different, even with identical levels of phosphorylation)
MTM
1. D2. Thve
3. Se(Sev
Phosphorylated forms can be separated by the amount and
site of phosphorylation.
12001200Phosphatase-treated time
Phosphorylated -casein
Non-phosphorylated -casein
Phosphorylated -casein+
Non-phosphorylated -casein
Conventional SDS-PAGE Phos-tag SDS-PAGE 10 % Acrylamide 10 % Acrylamide
100 M Mn2+-Phos-tagTM Acrylamide
Phos-tag SDS-PAGE and conventional SDS-PAGE was used to separate -casein samples, which have been dephosphorylated over time (incubation duration: 0120 min) by alkaline phosphatase.
Phos-tag ApplicationsMultiple possibilities of Phos-tag
Expressed Proteins Cells Animal tissues
Identification of new phosphoproteins
10. Related ProductsElectrophoresis/Western Blotting) P29
Phos-tag ApplicationsMultiple possibilities of Phos-tag
Expressed Proteins Cells Animal tissues
Identification of new phosphoproteins
Ready for research of phosphorylation at various stages and for various purposes
No need for applying radioactivity
Kinase Assay
Analysis of Kinase catalyzed signaling cascades.
Analysis of phosphorylation status of endogenous proteins
Increase / Decrease of phosphorylation by stimulation
Analysis of genetically modified mice
No need for preparation of Anti phospho-Antibodies
phos-tag.jp phos-tag.jp
2Phos-tag
SDS-PAGE is
2Phos-tag SDS-PAGE is Phos-tag SDS-PAGEPhos-tag SDS-PAGE is an electrophoresis technique capable of separating phosphorylated and non-phosphorylated forms based on phosphorylation levels. Various stains, Western blotting (WB) and mass spectrometry (MS) can be employed after electrophoresis. Phos-tag SDS-PAGE gels can be produced by adding a divalent metal (MnCl2 or ZnCl2) and Phos-tag Acrylamide (Phos-tag molecule bound to acrylamide) to the SDS-PAGE resolving gel.
Note
Prepared aqueous solution
Prepare with methanol or water
Product Name
Phos-tag Acrylamide
Phos-tag Acrylamide5 mM Aqueous Solution
Wako Cat. No.Nard Product #
300-93523AAL-107M
304-93521AAL-107
304-93526AAL-107S1
Pkg. Size
2 mg
0.3 mL0.9 mg
10 mg
Development from Phos-tag SDS-PAGEBy the combination of Phos-tag SDS-PAGE with various analysis methods, new information of phosphorylated proteins can be obtained.
Western blotting
Mass Analysis
Easy-recognizable phosphorylation of your target proteinsSimultaneous detection of phosphorylated/non-phosphorylated proteins with a general antibodyby their band shift differences.No need to prepare an antiphos-pho antibody.Applicable to analysis ofphosphorylation of endogenous proteins.
By separating phosphorylated forms, each phosphorylation site combination can be detected.
2D ElectrophoresisPhosphorylated forms with the same isoelectric point (same number of phosphorylation sites) can be separated. Application Data See the page #18
MC*ATP
Phosphorylated p35
Phos-tag SDS-PAGE
SampleRat brain extractDetectionAnti p35Lane 1brain extract before incubation.Lane 2-5Incubate with (+) and without (-) MC or ATP Data was provided byTomohisa Hosokawa at Brain ScienceInstitute, RIKEN (Japan)