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IMPORTANT: We have recently
(August 5, 2005) developed a new total GSK-3beta kit (Catalog No.
900-144) to replace our previous kit (Catalog No. 900-116). Our new kit
provides improved performance characteristics that will provide superior
results. The following Technical Bulletin outlines the differences
between the two kits:
total GSK-3beta Technical Bulletin
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| GSK 3beta
Colorimetric (EIA) Kits |
| Assay Format |
Catalog # |
Kit Insert |
MSDS |
Assay Layout |
| phospho: 96 Well Kit |
900-123 |
|
|
|
| total: 96 Well Kit |
900-144 (formerly
900-116) |
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FOR RESEARCH PURPOSES ONLY, NOT FOR DIAGNOSTIC USE. |
 
|
"A
Novel Quantitative Immunoassay for the Measurement of Total GSK-3beta"
(758 KB) - poster presented at ASCB 2003
Stable, Color-Coded Liquid Reagnets
Time to Answer: 3 Hours
|
Glycogen Synthase Kinase 3beta (GSK 3beta) is a unique serine/threonine
kinase that is inactivated by phosphorylation. In response to insulin
binding, PKB/AKT phosphorylates GSK 3beta on serine 9, which prevents
GSK 3beta from phosphorylating glycogen synthase. Unphosphorylated
glycogen synthase is active and able to synthesize glycogen. GSK 3beta
is also unique in that it requires a substrate that has been
phosphorylated by a distinct kinase before it can phosphorylate the
substrate. This phosphate priming mechanism explains why phosphorylation
of serine 9 inactivates GSK 3beta. The phosphorylated serine binds to
the GSK 3beta priming phosphate position and prevents binding of
alternative substrates. In addition to insulin signaling, GSK 3beta
participates in the Wnt signaling pathway, where it forms a complex with
axin, b-catenin and adenomatous polyposis coli (APC) protein. In the
presence of Wnts, GSK 3beta is unable to phosphorylate b-catenin, which
leads to stabilization of b-catenin. The Wnt pathway inactivates GSK
3beta via the proteins, Dishevelled and FRAT, which disrupt the
interaction of GSK 3beta with axin, beta-catenin, and APC. Clinically,
there is considerable interest in GSK 3beta inhibitors because they may
mimic the effect of insulin or reduce the hyperphosphorylation of Tau
that is observed in Alzheimer's Disease.
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1. Pipet Standards and Samples in duplicate into the wells.
2. Pipet the yellow Antibody into the wells and incubate.
3. Aspirate and wash the plate.
4. Pipet the blue Conjugate into the wells and incubate.
5. Aspirate and wash the plate.
6. Pipet the Substrate into the wells and incubate.
7. Pipet Stop Solution and read on a plate reader at 450 nm.
8. Calculate sample concentrations from Standard Curve.
|
| |
phospho GSK-3beta
(900-123) |
total GSK-3beta
(900-144) |
| RANGE |
62.5 - 2,000 pg/mL
|
78.1 - 5,000 pg/mL
|
| SAMPLE SIZE |
100 µL |
100 µL |
| SAMPLES PER 96
WELL KIT |
41 in duplicate
|
39 in duplicate
|
| SENSITIVITY |
24.1 pg/mL
|
74.4 pg/mL
|
| PRECISION |
2.7-11.6% Intra
6.3-11.4% Inter |
2.5-12.8% Intra
4.0-18.7% Inter |
SAMPLE TYPE
recommended dilution |
1 million Jurkat cells
per mL (1:8-32)
2 million Jurkat cells per mL (1:32-64)
4 million Jurkat cells per mL (1:64-128)
|
RIPA cell lysis buffer
2 ( 1:4)
2 million Jurkat cell/mL (1:6)
|
| TIME TO ANSWER |
3 Hours |
3 Hours |
| SPECIES
SPECIFICITY |
non-specific
|
non-specific
|
|
| Compound |
Cross Reactivity |
Compound |
Cross Reactivity |
| phospho
GSK-3beta |
| phospho GSK-3beta |
100% |
|
|
<0.01%: GSK-3alpha /
ATP Citrate Lyase / MEK / AKT / phosho JNK / ERK / phospho ERK
|
| total
GSK-3beta |
| total GSK-3beta |
100% |
|
|
| <0.01%: GSK-3alpha /
ATP Citrate Lyase / MEK 1 / AKT / phosho JNK / ERK / phospho ERK |
|
+32 (0) 16
58 90 45 
+32 (0) 16
50 90 45