CBA006 Sigma-AldrichPhosphoDetect™ ERK1/2 (pThr¹⁸⁵/pTyr¹⁸⁷) ELISA Kit

Figure 1: Effect of SDS Concentration in Cell Lysates on ERK1/2 [pTpY185/187] ELISA Signal

Lysates containing 10, 5, 2.5 and 1 mg/ml total protein from PMA-treated Jurkat cells were treated by the addition of 1, 0.5, 0.25 and 0.1% of SDS respectively. The cell lysates were then diluted 1:100, 1:50, 1:25, and 1:10 in standard diluent buffer to maintain a final concentration of 0.01% SDS. Upon dilution, equal aliquots of 10 µg total protein were loaded in the ELISA. The data shows that cell extracts with the added of 0.25-1% SDS generate higher signals than the extract with 0.1% SDS alone.

Figure 2: Effect of Protein Concentration on ERK1/2 [pTpY185/187] Recovery in Boiled Cell

Cell lysates containing total protein concentrations of 8, 5, 2, and 1 mg/ml were boiled for 5 min, centrifuged, and assayed (10 µg). The data shown show that protein concentration affects the signal of this ELISA, perhaps due to protein aggregation or precipitation after boiling

Figure 3: ERK1/2 (Total) and ERK1/2 [pTpY185/187] Sample Treatment Comparisons

Jurkat cells were treated with 100 ng/ml PMA for 10 min and cell lysates were prepared. Cell lysate prepared with Cell Lysis Buffer was either boiled for 5 min, treated with Sample Treatment Buffer, or not treated. Cell lysate was also prepared using Denaturing Cell Lysis Buffer. Samples were analyzed with the ERK1/2 and the PhosphoDetect™ ERK (Thr¹⁸⁵/Tyr¹⁸⁷) ELISA Kits.

Table 1: Dilution of ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷ Standard

Table 2: Intra-Assay Precision

Samples of known concentration of ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷ were assayed in replicates of 16 to determine precision within an assay.

Figure 4: Sensitivity

The sensitivity of this ELISA was compared to immunoblotting using known quantities of ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷. The data shows that the sensitivity of the ELISA is approximately 4X greater than that of immunoblotting. The bands shown in the immunoblotting were developed using rabbit anti-ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷ and an alkaline phosphatase conjugated anti-rabbit IgG followed by chemiluminescent detection.

Table 3: Inter-Assay Precision

Samples were assayed 48 times in multiple assays to determine precision between assays.

Table 4: Linearity

Jurkat cells were PMA-treated and lysed with Cell Lysis Buffer. This lysate was diluted in Standard Diluent Buffer over the range of the assay and measured for ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷ content. Linear regression analysis of sample values versus the expected concentration yielded a correlation coefficient of 0.99.

Figure 5: ERK1/2 (pThr¹⁸⁵/pTyr¹⁸⁷) ELISA: Parallelism

Natural ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷ from PMA-treated Jurkat cell lysates was serially diluted in Standard Diluent Buffer. The absorbance of each dilution was plotted against the ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷ standard curve. Parallelism is demonstrated in the figure above and indicates that the standard accurately reflects natural ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷ content in samples.

Figure 6: Peptide Competition

The specificity of this assay for dually phosphorylated ERK1/2 was confirmed by peptide competition. The data shows that only dual phospho-peptide containing the phosphorylated threonine and tyrosine could block the ELISA signal. The same sequence containing non-phosphorylated threonine and tyrosine at position 185/187 or mono-phospho peptides did not block the signal by more then 10%.

Figure 7: ERK1/2 (Total) and [pTpY185/187] Dose Response of PMA Stimulation on Jurkat Cell

Jurkat cells were treated with PMA at varying concentrations (0.1 to 1000 ng/ml) for 10 min, lysed and quantitated in parallel for ERK1/2 content (both ERK1/2 and ERK1/2 Phospho-Thr¹⁸⁵/Tyr¹⁸⁷). The amount of ERK1/2 remains constant, while the levels of phosphorylation at threonine 185 and tyrosine 187 increases with the dosage of PMA. The results correlate well with Immunoblot analysis of the same samples (see inset in the graph above).