① Optimization of the concentration of acrylamide
First, identify the optimum concentration of acrylamide＊ that allows migration of the target protein to the lowest end of the gel when conventional SDS-PAGE is used.
In Phos-tag™ PAGE, the migration speed is slower than in conventional SDS-PAGE (including non-phosphorylated proteins) and, therefore, the concentration of acrylamide should be examined (see the below figure). The migration speed decreases as the concentration of Phos-tag™ increases.
- more than 60 kDa: 6%; less than 60 kDa：8%
- ＜In case of high molecular weight proteins ＞
- The gel strength can be increased by adding agarose to gels that contain less than 4% of acrylamide. There is a data of separation of 350 kDa. (Refer to “Separation of Phos-tag™ Acrylamide” of 7. FAQ) Furthermore, the gel strength can also be enhanced by increasing the N,N'-methylenebisacrylamide content (eg: 5% acrylamide [24:1]).
② Optimization of the concentration of Phos-tag™ Acrylamide
Then, optimize the concentration of Phos-tag™ Acrylamide. Please evaluate the optimum concentration in the order of lowest to highest.
- 【Cell Lysate】
- In case there is a large variety of proteins in your sample, eg: cell lysates, the concentration of Phos-tag™ should be 5 to 25 μM. However, a higher concentration, eg: 100 μM, is recommended in case of a lower concentration of the target protein, eg: non-overexpression systems.
※The optimum condition depends on the protein. Please find the appropriate condition setting for each target protein.
（The information was provided by Yasunori Sugiyama at Science Research Cencer, Kochi University）
Higher concentration of Phos-tag™ leads to higher separation capacity?
In general, a higher concentration leads to higher separation capacity. (Compare the samples of 50 μM and 100 μM of Mn2+-Phos-tag™ in the left figure.) However, the higher concentration causes low velocity. It sometimes happens that the higher separation capacity is due to the lower Phos-tag™ concentration (Compare the samples of 50 μM and 150 μM of ovalbumin of the right figure.)