CBA069 Sigma-AldrichPhosphoDetect™ GSK-3β (pSer⁹) ELISA Kit

Table 1: Dilution of GSK-3β (pSer⁹) Standard

Table 2: Guidelines for diluting Anti-Rabbit IgG horseradish Peroxidase (HRP)

Table 3: Example Data

Example data obtained for the various diluted standards over the range of 0 to 100 Units/ml GSK-3β (pSer⁹).

Figure 1: Sensitivity

The sensitivity of this ELISA was compared to immunoblotting using known quantities of GSK-3β (pSer⁹). The data show that the sensitivity of the ELISA is approximately 2x greater than that of immunoblotting. The bands shown in the immunoblot data were developed an anti-GSK-3β (pSer⁹) and chemiluminescent detection.

Table 4: Intra-Assay Precision

Samples of known GSK-3β (pSer⁹) concentrations were assayed in replicates of 16 to determine precision within an assay.

Table 5: Inter-Assay Precision

Samples were assayed 36 times in multiple assays to determine precision between assays.

Figure 2: GSK-3β (pSer⁹) ELISA Parallelism

Lithium chloride-treated Jurkat cells were lysed and the lysate was serially diluted in Standard Diluent Buffer. The absorbance of each dilution was plotted against the GSK-3β (pSer⁹) standard curve. Parallelism is demonstrated by the figure and indicates that the standard accurately reflects GSK-3β (pSer⁹) content in samples.

Table 6: Linerearity of Dilution

Jurkat cells were grown in tissue culture medium containing 10% fetal calf serum were treated with 100 mM LiCl for 3 h and lysed with Cell Lysis Buffer. The lysate was diluted in Standard Diluent Buffer over the range of the assay and measured for GSK-3β (pSer⁹). Linear regression analysis of samples versus the expected concentration yielded a correlation coefficient of 0.99.

Figure 3: Peptide Blocking

The specificity of this assay for phosphorylated GSK-3β (pSer⁹) was confirmed by peptide competition. The data show that the phosphopeptide containing the phosphorylated Ser⁹ blocks the ELISA signal. Non-phosphorylated peptide does not block the signal.

Figure 4: GSK-3β (pSer⁹] and GSK-3β

Jurkat cells were treated with 100 mM LiCl for 2 h and untreated Jurkat cells were used as control. Cell extracts were prepared in 0.1% SDS extraction buffer, and analyzed using the GSK-3β (pSer⁹) ELISA and GSK-3β ELISA. The results show that the phosphorylation of GSK-3β (pSer⁹) is upregulated in LiCl-treated Jurkat cells, whereas the level of total GSK-3β remains consistant in LiCl-treated samples and untreated control.