Chemiluminescent reporter gene assays: sensitive detection of the GUS and SEAP gene products.
Chemiluminescent assays are described for the secreted alkaline phosphatase (SEAP) and beta-glucuronidase (GUS) reporter gene products. These assays provide simple, sensitive, non-isotopic alternatives to existing detection methods and are performed in microplate or tube luminometers or in a scintillation counter. The SEAP reporter gene product is secreted from mammalian cells and is thus easily detected in a sample of culture medium. Sensitive detection of secreted placental alkaline phosphatase is performed with CSPD chemiluminescent alkaline phosphatase substrate, and approximately 3 fg of enzyme can be detected. GUS has become the major reporter gene used for the analysis of plant gene expression. Sensitive chemiluminescent detection of GUS activity can be performed with an assay system we have developed using Glucuron, a beta-glucuronidase substrate. This chemiluminescent assay detects 60 fg of GUS and is linear over six orders of magnitude of enzyme concentration. CSPD and Glucuron substrates have been incorporated into two new chemiluminescent reporter gene assay kits for SEAP and GUS.
Bronstein
I
I
Tropix, Inc., Bedford, MA.
Fortin
J J
JJ
Voyta
J C
JC
Juo
R R
RR
Edwards
B
B
Olesen
C E
CE
Lijam
N
N
Kricka
L J
LJ
D000469:Alkaline Phosphatase / Q000032:analysis* / Q000235:genetics; D058851:GPI-Linked Proteins; D017930:Genes, Reporter; D005966:Glucuronidase / Q000032:analysis* / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000032:analysis* / Q000235:genetics; D008163:Luminescent Measurements; D010920:Placenta / Q000201:enzymology*
D058851:GPI-Linked Proteins; D007527:Isoenzymes; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental; D005966:Glucuronidase