Phos-tag™ PAGE GUIDEBOOK

Phos-tag™ SDS-PAGE is …

Phos-tag™ SDS-PAGE

Phos-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.

Features

  • It can be used regardless of the type and position of amino acid residues.

  • ・It can be used in phosphorylation analysis of unknown phosphoproteins.
  • ・It can be used in the detection of new phosphorylation sites.
  • Phosphorylated forms with different amounts and location of phosphorylation sites can be separated.

  • ・Able to determine the level of phosphorylation, as well as the amount of phosphorylated forms.
  • Capable of simultaneously detecting phosphorylated and non-phosphorylated forms.

  • ・Able to quantify various phosphorylated forms.
  • ・Easily determine the presence of phosphorylation
  • Radioisotopes or special equipment is not necessary (can be used immediately if there are SDS-PAGE reagents and equipment)

  • ・Experiments can be carried out easily and at low costs.
  • After electrophoresis, WB- and MS-based analysis, as well as 2D gel electrophoresis can be performed.

  • WB: internal control protein analysis is possible. (See Application Data 3 and 4.)
  • MS:phosphorylation site combinations of various phosphorylated forms can be determined. (See Application Data 1 and 2.)
  • 2D gel electrophoresis:phosphorylated forms with identical isoelectric points or molecular weights can be separated. (See Application Data 2.)

Development from Phos-tag™ SDS-PAGE

By the combination of Phos-tag™ SDS-PAGE with various analysis methods, new information of phosphorylated proteins can be obtained.

Western blotting

Easy-recognizable phosphorylation of your target proteins
Simultaneous detection of phosphorylated/non-phosphorylated proteins with a general antibody by their band shift differences.
No need to prepare an antiphospho antibody.
Applicable to analysis of phosphorylation of endogenous proteins.

Western blotting

Mass Analysis

By separating phosphorylated forms, each phosphorylation site combination can be detected.

2D Electrophoresis

Phosphorylated forms with the same isoelectric point (same number of phosphorylation sites) can be separated.
Application Data

Phos-tag™

Application using Phos-tag™ SDS-PAGE

related products

Objective Sample type Application Related Products Application Data
To identify phosphorylation sites Lysate Ala substitute + WB ImmunoStar Series Application Data
Purified protein Recombinant + MS
Immunoprecipitation
+ MS
Silver Stain MS Kit,
nanoLC-MS/MS
Application Data ,
To simultaneously detect and quantify various phosphorylated forms Lysate WB ImmunoStar Series Application Data
,,
Purified protein CBB staining, Silver
staining, etc.
Quick CBB Plus,
Silver Stain MS Kit, etc.
Application Data ,
To confirm presence of phosphorylation Lysate WB ImmunoStar Series Application Data
,,
To perform additional separation Immunoprecipi-
tation sample
2D Electrophoresis Quick CBB Plus,
Silver Stain MS Kit, etc.
Application Data
To search for kinase or inhibitors effective against the target protein Lysate Column Chromato-
graphy + WB
ImmunoStar Series Application Data
Purified protein Recombinant kinase
+ CBB staining,
Silver staining, etc.
-
Application Data
Product Name Pkg. Size Wako Cat. No.
(Nard Product #)
Note
Phos-tag™ Acrylamide 5 mM Aqueous Solution 0.3 mL
(0.9 ㎎)
304-93526 (AAL-107S1) Prepared aqueous solution
Phos-tag™ Acrylamide 2 ㎎ 300-93523 (AAL-107M) Prepare with methanol or water
10 ㎎ 304-93521 (AAL-107)

Important differences compared to conventional SDS-PAGE and cautions

There are some important differences between Phos-tag™ SDS-PAGE and conventional SDS-PAGE that should be considered.

  • ◆ Prior sample preparation is required.

  • Due to Phos-tag™ SDS-PAGE being easily influenced by EDTA, prior sample processing (such as TCA precipitation) is highly recommended⇒Details.
  • ◆ Migration velocity is slower

  • Even with non-phosphorylated proteins, the migration velocity in Phos-tag™ SDS-PAGE is lower than in conventional SDS-PAGE.⇒Details.
  • ◆ Unable to perform molecular weight estimations using molecular weight markers.

  • In Phos-tag™ SDS-PAGE, molecular weight estimations using molecular weight markers is not possible. Please simply use the markers as a guide of WB transfer efficiency. ⇒Details. Additionally, pre-stained markers can cause band distortions. It is recommended to use a recombinant or dephosphorylated sample of the target protein as a marker.⇒Details.
  • ◆ EDTA treatment is required for WB transfer.

  • In order to increase the transfer efficiency of Phos-tag™ SDS-PAGE to WB, EDTA treatment is required prior to transfer.
  • ◆ When performing Phos-tag™ SDS-PAGE, please concurrently perform a conventional SDS-PAGE as a control.

  • This is required when multiple bands are detected in Phos-tag™ SDS-PAGE, in order to determine whether the target protein is phosphorylated or degraded.