In Situ Detection Method for Glutaminyl Cyclase Activity in Polyacrylamide Gels

that achieved by TLC with numerous buffers (1,2,5) or by HV-TLE protocols (2) when multiple samples are analyzed. We have found that when 1 μL of a dye solution (1% orange G, 1% phenol red in pH 3.5 buffer) was electrophoresed in parallel with the phosphoamino acid standards, orange G migrated slightly ahead of pSer and phenol red migrated with pTyr, providing useful visible markers for electrophoretic progression in our system. When proteins are labeled by in vitro kinase reactions, the only radioactive compounds in the hydrolysates will be phosphopeptides, phosphoamino acids and phosphate. Under these conditions, a one-dimensional separation by TLE at pH 3.5 is considered adequate (2). When proteins are labeled in vivo and only partially purified, a high resolution two-dimensional HV-TLE/TLC separation system is required. These hydrolysates frequently contain labeled compounds that migrate with the phosphoamino acids (2). The procedure we describe is therefore limited to PAA of purified proteins and substrates labeled in vitro.