Phos-tag™ PAGE GUIDEBOOK

Protocol

Two Phos-tag™ SDS-PAGEs

Depending on the type of divalent metal ion that binds to the Phos-tag™ molecule and the composition of buffers used in gel preparation, Phos-tag™ SDS-PAGE gels can be separated into two types as shown below. Since there are various features available, please choose accordingly to your needs. Various examples of usage are described here.

Mn2+-Phos-tag™ SDS-PAGE  GelApplication

Zn2+-Phos-tag™ SDS-PAGE GelApplication1

Gel Type Advantages Disadvantages
Mn2+-Phos-tag™
SDS-PAGE
・Protocol similar to Laemmli method
・Semi-dry transfer possible even with high concentrations of Phos-tag™
・Some proteins are in separable.
・Gel preparation should be just before use.
Zn2+-Phos-tag™
SDS-PAGE
・High resolving capability
・Ability to separate a wide range of proteins and phosphorylated forms
・Long storage life of gels
・Semi-dry transfer rate is poor at high concentrations of Phos-tag™
・Bands will smear in low concentration gels with less than 5%.
Basic Structure of Phos-tag™
80μM Phos-tag™ Acrylamide, 7.5 % Polyacrylamide gel
Detection : Fluorescent staining

In Zn2+-Phos-tag™ SDS-PAGE

  • ・Resolving capabilities were improved.(ABL, MET)
  • ・Bands unable to be separated on Mn2+-Phos-tag™
    SDS-PAGE could be resolved. (FYN)
  • [Sample of each lane]
    Left : non-phosphorylated Tau
    Right : phosphorylated Tau
    (phosphorylated form by ABL, MET, FYN)
  • Improved Phos-tag SDS-PAGE under neutral pH conditions for advanced protein phosphorylation profiling.
    E Kinoshita and E Kinoshita-Kikuta, Proteomics, Jan 2011; 11(2): 319-23.

Mn2+-Phos-tag™ SDS-PAGE

*1) For the MnCl2 solution, two times the Phos-tag™ concentration(molar ratio) is added.

*2) The conventional concentration can be used for TEMED and Sol.G. The above added amounts are examples.

*3) The normally used concentration can be used for TEMED and Sol.G. The above added amounts are examples.

*4) Add the agarose after distilled water has been added and thoroughly dissolved in a microwave oven and it is still hot.

*5) If necessary, preheat the pipette tip and gel preparation table to 40 - 45℃.

Zn2+-Phos-tag™ SDS-PAGE

Though the Mn2+-Phos-tag™ SDS-PAGE adopts the typical Laemmli SDS-PAGE method and is a simple procedure, there have been cases where separation of phosphorylated and non-phosphorylated forms was not possible, depending on the protein. In contrast, Zn2+-Phos-tag™ SDS-PAGE using the neutral Bis-Tris gel SDS-PAGE system dramatically improves resolution, since Phos-tag™ shows the highest phosphoric acid group-capturing ability at neutral pH.

<Separation of large phosphorylated proteins bigger than 200kDa>

In conventional Zn2+-Phos-tag™ SDS-PAGE, Bis-Tris is used in the resolving and stacking gels. Bis-Tris acts as a radical quencher since its structure is similar to TEMED. As a result, the bands smear due to a lost in sieving effect in low concentration polyacrylamide gels (less than 5%). If performing Zn2+-Phos-tag™ SDS-PAGE using 3% or 4% low concentration polyacrylamide, please prepare gels with Tris-AcOH in place of Bis-Tris. Please refer to【1】-③ as well. Recommended for large proteins above 200kDa.

Composition Tris-AcOH gel Bis-Tris gel
Running buffer ・50mM Tris ・0.1%(w/v) SDS
・50mM Tricine ・5.0mM Sodium Bisulfite
・100mM Tris ・0.1%(w/v) SDS
・100mM MOPS ・5.0mM Sodium Bisulfite
Resolving / Stacking buffer ・200mM Tris-AcOH (pH 7.0) ・357mM Bis-Tris-HCl (pH 6.8)
Sample buffer ・Conventional Laemmli System ・Conventional Laemmli System
Gel concentration ・3〜4 % Polyacrylamide + 0.5 % Agarose ・≧ 5 % Polyacrylamide

Post-Phos-tag™ SDS-PAGE analysis

◆ Western Blotting
vvv
When transferring phosphorylated forms from Phos-tag™ SDS-PAGE gel to the membrane, transferring rate tends to decrease considerably. In order to improve the transfer rate, it is necessary to remove Mn2+ and Zn2+ using EDTA. Please carry out the following depending on the type of transfer apparatus used.

① Using the semi-dry method

  • (1) After electrophoresis, please immerse the gel in transfer buffer containing 10 mmol/L EDTA and shake gently.
    (1 to 3 times for 10 min each)
    ※Here is an example of EDTA concentration, processing time, frequency of EDTA-containing buffer changing.
    Please optimize conditions as necessary.
    ※Please change the EDTA-containing buffer depending on gel thickness. (Example: 1.5 mm thickness: 2 times for 20 min)

  • (2) Next, please immerse the gel in transfer buffer without EDTA.

Phos-tag™ SDS-PAGE

ATTENTION
When using highly concentrated Zn2+-Phos-tag™ gels (example: 100 μM Phos-tag™), sufficient transfer rate may not be attained even after EDTA treatment. If such is the case, please use the tank method.

② Using the tank method (wet)

Please use transfer buffer containing 0.1% SDS. When using the tank method, the EDTA treatment step can be omitted as long as SDS-containing transfer buffer is used. Pay careful attention as proteins may fall off the membrane. Please perform the procedures using SDS at the optimal concentration of 0.05% to 0.2%

Wako Cat. No. 019-25111 AquaBlot™ 10 × Tris-Glycine-
SDS Transfer Buffer (1L)

◆ Quantification analysis

Please perform silver staining and Coomassie Brilliant Blue staining after electrophoresis, and then follow the usual in-gel digestion protocol to prepare samples. EDTA treatment procedures are not necessary.

Wako Cat. No. 299-58901 Silver Stain MS kit(20 tests)