phos-tag™ sds-page guidebook - wako-chem.co.jp .compound designed from an alkaline phosphatase

Download Phos-tag™ SDS-PAGE GUIDEBOOK - wako-chem.co.jp .compound designed from an alkaline phosphatase

If you can't read please download the document

Post on 07-Jul-2018

221 views

Category:

Documents

2 download

Embed Size (px)

TRANSCRIPT

  • 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)