Cell Signaling ELISA Kits and Multiplex Immunoassays

Figure 2: Measurement of Total vs. phosphorylated AKT in Jurkat cells using AKT ELISA. Jurkat cells were treated with increasing concentrations (0–500 nM) of wortmannin (a PI-3 kinase inhibitor). Cells were lysed and assayed in parallel for AKT [Total] and phosphorylated AKT [pS473] using AKT (Total) Human ELISA Kit (Cat. No. KHO0101) and AKT (Phospho) [pS473] Human ELISA Kit (Cat. No. KHO0111) ,respectively. The graph shows that the amount of total AKT remained comparable, however the levels of phosphorylation at serine 473 decreased in a dose-dependent manner with increase in wortmannin.

Figure 3. Specificity of AKT [pS473] ELISA. Peptide competition assay was conducted to confirm the specificity of AKT [pS473]. Titration of a specific blocking peptide to phosphorylation of AKT [pS473], was measured using AKT (Phospho) [pS473] Human ELISA Kit (Cat. No. KHO0111). The graph shows that only the peptide corresponding to the region surrounding serine 473 blocks the ELISA signal.

Figure 4. Analytical sensitivity of AKT [pS473] ELISA. Jurkat cells were cultured under optimal conditions and phosphorylated AKT cell extracts were obtained. Stimulation of phosphorylated AKT [pS473] was measured in cell lysate using AKT (Phospho) [pS473] Human ELISA Kit (Cat. No. KHO0111) and Western blot. The analytical sensitivity of AKT [pS473] ELISA was determined by adding two standard deviations to the mean O.D. obtained from 30 assays of the zero standard. The value of <0.8 Units/mL corresponds to the amount of AKT [pS473] extracted from 4000 Jurkat cells. Results showed that the sensitivity of the ELISA is ~2-fold greater than that of western blot when tested against known quantities of AKT [pS473].

Figure 5. Detection of human p38 MAPK [pT180/pY182] in HEK293 cells using InstantOne ELISA. HEK293 cells were treated with 2 µg/mL of anisomycin (a potent activator of p38/JNK MAPK pathway) for 30 min. Untreated HEK293 cells (-) served as negative control. These cells were analyzed by Western Blot (right) and ELISA (left) using p38 MAPK (Phospho) [pT180/pY182] Multispecies InstantOne ELISA Kit (Cat. No. 85-86022-11). The intensity of the band at 38 kDa as detected by immunoblot correlates with InstantOne ELISA absorption values.

Figure 6. Detection of SMAD1 (Total/Phospho) in C2C12 cells using InstantOne ELISA. C2C12 cells were either inhibited with dosomorphin (50 mM) for 60 min) (-) or stimulated with BMP4 (10 ng/mL) for 30 min (+). Lysates were prepared as described in the InstantOne ELISA user manual. Cells (10 µg/assay) were analyzed for total SMAD1 (left) or phosphorylated SMAD1 [S463/S465] (right) using SMAD1 (Total/Phospho) InstantOne ELISA Kit (Cat. No. 85-86183-11).

A) For ProcartaPlex assays the Luminex beads are coupled with capture antibodies that specifically bind to signaling proteins, regardless of their phosphorylation state. The conjugation of a specific antibody to a distinct bead number enables the analysis of multiple analytes in a single well. Separate panels are created for multiplex analysis of total or phosphorylated signaling proteins using non-phospho-specific or phospho-specific detection antibodies with PE-dye for reporter channel 1 (532nm), making them compatible with all Luminex instruments.

(B) For ProcartaPlex Dual Reporter assays the same capture antibodies are applied, but for quantitation of phosphorylated signaling proteins a phospho-specific detection antibody with BV421-conjugation is used that leverages the additional reporter channel 2 (405 nm) of the Luminex xMAP INTELLIFLEX DR-SE instrument. Total proteins are detected through reporter channel 1 (532 nm) with the PE dye. This approach enables the simultaneous quantitation of both total and phosphorylated proteins in a single well on the same bead.

Figure 7. Comparison data to demonstrate correlation of ProcartaPlex signaling assays with conventional ELISA. Lysates of various cell lines were used in duplicates and run in parallel on ProcartaPlex Signaling Simplex kits (Cat. No. EPX010-97101-PHO, Cat. No. EPX010-97106-PHO,  Cat. No. EPX010-97102-PHO) and ELISA kits (Cat. No. KHO0111, Cat. No. KHO0421, Cat. No. KHO0241). Presented data of the measured concentrations of Akt[pS473], PRAS40[pT246] and CREB[pS133] show high correlation (R2 > 0.8). This demonstrates that ProcartaPlex Signaling Simplex kits achieve quantitative results comparable to traditional ELISA kits with the additional benefits of multiplexing including reduced sample volume, shorter assay/hands-on time and lower costs per data point. 

Figure 8. Comparison data for ProcartaPlex and western blot. Performance comparison of protein analysis using western blot and ProcartaPlex Human Akt Pathway Dual Reporter Panel, 2x8plex (Cat. No. EPX080-97200-DR) demonstrates a high correlation between the two assay systems. Western blot data shows PRAS40[pT246] levels (40 kDa) in lysed NIH-3T3 cells. Equal amounts of protein (10µg) were used for western blot and ProcartaPlex assays. ProcartaPlex Human Akt Pathway Dual Reporter Panel, 2x8plex (Cat. No. EPX080-97200-DR) was performed following the user guide. For western blot analysis, protein samples were electrophoresed using Bolt 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gels 17slots (Cat. No. NW04127BOX), XCell SureLock Electrophoresis System (Cat. No. EI0002) and PageRulerPlus Prestained Protein Ladder, 10 to 250 kDa (Cat. No. 26619). Resolved proteins were then transferred onto a nitrocellulose membrane iBlot 2 Transfer Stacks, nitrocellulose (Cat. No. IB23002), with iBlot 2 Dry Blotting System. The membrane was probed with the relevant primary and secondary antibody after blocking using iBind Solution Kit (Cat. No. SLF1020). Chemiluminescent detection was performed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Cat. No. 34577)

Figure 9.Comparison of analytical sensitivity of ProcartaPlex Signaling Assay and western blot. Jurkat cells were cultured under optimal growing condition and cell lysate were used to measure phosphorylated CREB. Same amount (10 µg) of proteins were used for a serial 1:2 dilution and measured with ProcartaPlex CREB[pS133] Simplex kit (Cat. No. EPX010-97102-PHO) and western blot assay. For western blot, the same phospho-specific detection antibody against PRAS40[pT246] was used as included in the Simplex kit. Lowest concentration measured with western blot was 5 µg/well Lowest concentration measured with ProcartaPlex Simplex kit was 0.6 µg/well.

Figure 10. Correlation between conventional ProcartaPlex and ProcartaPlex Dual Reporter format. ProcartaPlex Human Akt Pathway Dual Reporter Panel, 2x8plex (Cat. No. EPX080-97200-DR) was utilized alongside the ProcartaPlex Human Akt Pathway Panel (total), 8plex (Cat. No. EPX080-97000-901) and ProcartaPlex Human Akt Pathway Panel (phospho), 8plex (Cat. No. EPX080-97100-PHO) to assess total or phosphorylated proteins involved in the Akt signaling pathway. The obtained data demonstrate a strong correlation between the Dual Reporter Panel and the conventional panels for either total or phosphorylated proteins (R² > 0.9).