Trouble Shooting

Introduction ‒ Key to success is "preparation of samples" ‒

The sample condition makes a significant difference. Prepare samples keeping the following points in mind:

  • ● Contamination of influential substances: In particular, never use EDTA. Make sure that commercially available inhibitor cocktails and buffers do not contain EDTA. Also eliminate EDTA from constituents of media.

  • ●Sample condition: Do not use highly viscous samples such as overconfluent cell lysates (dilution does not improve much).

  • ● Standardization of buffer composition for samples: Standardize the composition of the buffer for each sample to be applied to the gel. Pay particular attention when samples and markers treated with phosphatase are used. Apart from chelating agents and surfactants, concentrations of MnCl2 and ZnCl2 have some influences.

  • If the preparation has a trouble, it is advised to re-prepare the sample paying attention to the above.

Popular trouble shootings (causes and actions taken)

Distortion of bands

smearing of bands

Basic Structure of Phos-tag™
① No EDTA treating
② 1mM EDTA for 10 minutes x 2 times
③ 10mM EDTA for 10 minutes x 1 times
④ 1mM EDTA for 10 minutes x 2 times

【Sample】 WIDE-VIEW™ Prestained protein Size Marker Ⅲ(Wako Cat. No.230-02461, etc.)

【Gel】SuperSep™ Phos-tag™ (50μmol/L), 12.5%, 13 wells (wako Cat. No. 196-16701)

  • * The transfer rate decreases considerably if EDTA treatment is not performed (by comparison of ① with ②, ③ and ④). First of all, It is advised to wash twice with 1-10 mM EDTA each for 10 minutes. In particular, duration of the treatment and number of exchanges of EDTA-containing buffer are important.

Low resolution

Also consider the following methods.

  • • Increase the molar ratio of metal ion (MnCl2 or ZnCl2) to Phos-tag™ acrylamide in the gel (e.g. 1:4).

  • • Use Tris-Tricine buffer as the running buffer.

  • (In the case of Mn2+-Phos-tag™ SDS-PAGE) Consider Zn2+-Phos-tag™ SDS-PAGE.

  • • Prepare the reagents freshly.

Wako Cat. No. 200-17071 Tricine Running Buffer
Solution(x10)(1 L)

Other trouble shootings

  • Phosphorylated form is not obtained from immnunoprecipitation samples.
    When a monoclonal antibody is used, a phosphorylated form may not be obtained due to overlapping of the epitope and the phosphorylated region. For purification of the target protein by the immnunoprecipitation, it is advised to use a polyclonal antibody.

  • The sample condition is unusual.
    Washing with PBS in the preparation of cell lysate may affect the phosphorylation condition. Add TCA immediately after elimination of the medium.

  • It is difficult to judge whether the gel or the sample is problematic.
    A mixture of phosphorylated α-casein and dephosphorylated α-casein is available as a positive control. Perform the ordinary SDS-PAGE simultaneously using this product as a sample.

  • Phos-tag™ acrylamide does not dissolve.
    Warming at about 40℃ or treatment with an ultrasonic bath after addition of methanol and water makes dissolution easier.

  • Separation and migration speed vary among lanes.
    The concentration of the substance interfering with Phos-tag™ (such as EDTA, Mn2+, Zn2+) may differ considerably depending on the sample. Prepare samples taking care not to vary the concentration among them.

Protein Diffusion

Long-term migration with a constant current will cause decomposition and diffusion of proteins due to excessive heat.

  • ① If you want to use a constant current for migration, try techniques such as using a low-temperature room, thoroughly cooling the migration buffer just before use, and wrapping a cooling agent around the migration tank (but do not use ice because it may cause electric shock).

  • ② When a constant voltage can also be used, migrate with a constant voltage (eg: 200 V). The migration speed will slow down but the generation of heat will be suppressed.

Easy breaking of the gel

The gel is softened due to the low concentration of acrylamide.

  • ① 5% or higher:Increasing the N,N’-methylene-bisacrylamide to acrylamide ratio (eg: 24:1) will strengthen the gel.

  • ② Add 3〜5% of agarose to strengthen gels. Refer to “Preparing a Low-Concentration Gel Containing Agarose” in “Protocol” and “Separation” in “FAQ.”

The background becomes high after staining.

Carry out staining after eliminating metal ions in the gel by EDTA treatment similarly to the Western blotting.