At first, 10 μM N-His6-PhoB with 16 extra amino acids, 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 10 mM MgCl2 with 50 mM lithium potassium acetyl phosphate were reacted at 37℃, remained 90 min. After that, I added SDS loading buffer and heated it at 95℃ for 10 min. Then, Phos-tag SDS-PAGE included 50 μM Phos-tag acrylamide and 100 μM MnCl2 was used to separation at 30 mA for 60 min. But, only one protein band identical to the control is there.

From the research of professor Eiji Kinoshita and professor Ann M. Stock, I have learn that the phospho-Asp is not stable and easily hydrolysis. So the heating after the add of SDS loading buffer and trichloroacetic acid (TCA) precipitation method are not suitable. So, I try the method of professor Ann M. Stock, not heat and not treat it with TCA method before loading on gel. And changing the condition from 30mA to 150V to reduce the heat production. However, only one protein band is there. And the sample with acetyl phosphate even run faster than the control without acetyl phosphate. But some difference exists here. They used a chemical synthetic ammonium hydrogen phosphoramidate (NH2-PO3) believed as a more efficient reagent but not acetyl phosphate (CH3-CO-O-PO3). And they used PhoB-C-thrombin-His6 and treated it with thrombin.

In these experiments, whatever treated with TCA method and heated or not, α-casein can be separated. Based on this result, one possible is PhoB haven’t been phosphorylated, another is incorrect operation in Phos-tag SDS-PAGE. But I have no idea.

In the protein purification process, I’m not sure N-His-tag PhoB with 16 extra amino acids is okay or not. Previously, I store the protein PhoB in 50 mM Tris-HCl, pH 8, 100 mM NaCl and 20% glycerol. Is that influence?

In the reaction process, 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM 2-mercaptoethanol, 10 mM MgCl2 with 50 mM lithium potassium acetyl phosphate were reacted at 37℃, remained 90 min. I want to know if it suitable or not. And I found that there was a white insoluble substance in the experiment group, but not in the control group without lithium potassium acetyl phosphate. Why and does this have some influence? And I’m not sure the purity of acetyl phosphate (≥97.0% NT) is ok or not, some research used more expensive acetyl phosphate A0262 (≥85% elemental analysis) produced by Sigma-Aldrich but not the 01409 (≥97.0% NT).

After reaction, I find many research usually end the reaction by adding SDS loading buffer. Based on my experiment, I think acetyl phosphate will influence the effect of Phos-tag. But I’m not sure when and how to remove acetyl phosphate. And I not sure the ammonium hydrogen phosphoramidate (NH2-PO3) also have influence or not?

Now, I plan to repeat the phosphorylation experiment start from PhoB purification. So, I want to try my best to correct the wrong process and operate with a correct protocol. But I don't know what the key issue was that caused the experiment to fail. I pretty hope to get some direction, and it will be a great help for me and my research. Looking forward to hearing from you! Thanks a million!