SEAP Chemiluminescence Kit 2.0
SEAP Chemiluminescence Kit 2.0
Brand: Takara Bio.
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SEAP Chemiluminescence Kit 2.0
The Great EscAPe SEAP Chemiluminescence Kit 2.0 is a complete system for the detection of secreted alkaline phosphatase (SEAP) activity in the growth media from mammalian cells expressing this enzyme. The kit contains reagents necessary for the chemiluminescent detection of SEAP activity using a 96-well plate luminometer. The assay can also be performed in single tubes and measured in a tube luminometer. It allows you to monitor promoter activity over time, because samples of the cell media can be taken repeatedly and tested without lysing cells.
Overview
- One step assay, for monitoring promoter activity
- 10-fold higher sensitivity then firefly luciferase
- No-cell-lysis protocol
- Adaptable from single-tube to high-throughput assays
- Dual, live-cell assay includes secreted luciferase as well as SEAP:
monitor two promoters, or use one reporter as a normalization control
Applications
- Time-course studies
- Non-invasive reporter gene assays with secreted alkaline phosphatase
- Test multiple compounds
- Perform downstream experiments with the same cells
- Use the dual assay (which includes secreted luciferase, as well a secreted alkaline phosphatase assay) to monitor two promoters
- Use the dual assay to monitor one promoter with a built-in normalization control
Chemiluminescent detection of protein-protein interactions
Chemiluminescent detection of protein-protein interactions. The bait vector pM-53 expresses p53 fused to the GAL4 DNA-binding domain. When HEK 293 cells were cotransfected with this bait together with the SV40 large T antigen prey vector, pVP16-T, strong expression of SEAP was detected because large T antigen interacts with p53. The CP protein, which does not interact strongly with p53, was used as a negative control.
SEAP activity is highly specific, with a >20-fold increase in activity
SEAP activity is highly specific, with a >20-fold increase in activity. HEK 293 cells were transiently transfected with a promoter construct containing CRE driving the expression of SEAP, or mock-transfected. 12 hr posttransfection, the cell culture media was replaced by media containing either 10 μM forskolin or plain cell culture media and incubated for 7 hr. Forskolin causes an increase in the level of cytosolic cAMP, which in turn activates CRE, driving the expression of SEAP, which is detectable in the media culture supernatant. The samples were assayed using the Great EscAPe SEAP Chemiluminescence Kit 2.0 and its protocol and analyzed on a BD Monolight 3096 Luminometer.
Comparison of the sensitivities of SEAP and firefly luciferase
Comparison of the sensitivities of SEAP and firefly luciferase. Parallel cultures of BHK cells were transiently transfected with the indicated amounts of either pSEAP2-Control Vector or a similar vector expressing luciferase. After 24 hr, SEAP activity was assayed in the appropriate culture media using the Great EscAPe chemiluminescent assay. Similarly, after 24 hr, cell lysates were prepared from the luciferase cultures, and luciferase activity was assayed using a commercial kit. RLU = relative light units.
Signal transduction kits are available for in vivo studies of signal transduction—from signaling cascades to gene transcription
Signal transduction kits are available for in vivo studies of signal transduction—from signaling cascades to gene transcription.
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Chemiluminescent detection of protein-protein interactions
Chemiluminescent detection of protein-protein interactions. The bait vector pM-53 expresses p53 fused to the GAL4 DNA-binding domain. When HEK 293 cells were cotransfected with this bait together with the SV40 large T antigen prey vector, pVP16-T, strong expression of SEAP was detected because large T antigen interacts with p53. The CP protein, which does not interact strongly with p53, was used as a negative control.
SEAP activity is highly specific, with a >20-fold increase in activity
SEAP activity is highly specific, with a >20-fold increase in activity. HEK 293 cells were transiently transfected with a promoter construct containing CRE driving the expression of SEAP, or mock-transfected. 12 hr posttransfection, the cell culture media was replaced by media containing either 10 μM forskolin or plain cell culture media and incubated for 7 hr. Forskolin causes an increase in the level of cytosolic cAMP, which in turn activates CRE, driving the expression of SEAP, which is detectable in the media culture supernatant. The samples were assayed using the Great EscAPe SEAP Chemiluminescence Kit 2.0 and its protocol and analyzed on a BD Monolight 3096 Luminometer.
Comparison of the sensitivities of SEAP and firefly luciferase
Comparison of the sensitivities of SEAP and firefly luciferase. Parallel cultures of BHK cells were transiently transfected with the indicated amounts of either pSEAP2-Control Vector or a similar vector expressing luciferase. After 24 hr, SEAP activity was assayed in the appropriate culture media using the Great EscAPe chemiluminescent assay. Similarly, after 24 hr, cell lysates were prepared from the luciferase cultures, and luciferase activity was assayed using a commercial kit. RLU = relative light units.
Signal transduction kits are available for in vivo studies of signal transduction—from signaling cascades to gene transcription
Signal transduction kits are available for in vivo studies of signal transduction—from signaling cascades to gene transcription.
