In our lab, we use secreted alkaline phosphatase (SEAP) as a positive control for protein trafficking in conjunction with oocyte voltage clamping experiments. As we searched for a SEAP assay, we wanted to find one that was fast and simple but most importantly, one that was precise. Applied Biosystems’ Phospha-Light System for detecting SEAP fit the bill.
This assay relies on a chemiluminescent reporter gene system to detect SEAP in culture media in about an hour. Additionally, non-secreted alkaline phosphatase can be detected in both cell and tissue extracts. Protocols for detecting both secreted and non-secreted alkaline phosphatase come with the kit. Also advantageous are the lack of complicated buffers to make and the inclusion of everything needed for the assay (except RO water).
The actual procedure is also fairly simple. The evening before the assay, I placed 10 frog oocytes per treatment in a 96-well plate (one oocyte/well) with 100 uL of media. Each oocyte had been injected with RNA encoding proteins involved in sodium transport. The experimental group of oocytes was additionally injected with RNA that encoded a protein thought to inhibit sodium transport. After incubating the oocytes for about 18 hours, I removed 50 uL of conditioned media and added 150 uL of dilution buffer in a 0.5 mL PCR tube. The sample was then incubated at 65oC for 30 minutes, followed by a rapid cool down to room temperature. Next, I removed 50 uL of each sample from its respective tube and placed it in a well of an opaque 96-well plate. To each well, I added 50 uL of assay buffer and incubated at room temperature for five minutes. Lastly, I added 50 uL of reaction buffer and incubated the samples for 20 min at room temperature. Following this final incubation, I placed the plate in a luminometer and measured the chemiluminescense in each well. We found that there was a significantly lower amount of SEAP in the oocytes co-injected with a putative inhibitor than in the control group. However, the amount of alkaline phosphatase inside the oocytes was not different. This indicated to us that our inhibitor was not specific to our protein of interest but rather had a more general affect on exocytosis.
The only real problem I have encountered while using this kit is the paucity of technical assistance. I called with a question regarding lysis solution compatibility in preparation for measuring non-secreted alkaline phosphatase, but was only able to leave a message and my question still remains unanswered. Other than that, I would recommend this kit to anyone needing a quick and inexpensive way to measure SEAP.
J. Jason Clark
Research Assistant
University of Iowa