HTRF Human Total SKP2 Detection Kit, 10,000 assay points

Total SKP2 assay principle

The Total SKP2 assay measures SKP2 levels in cells. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. Both antibodies are highly specific for a distinct epitope on the protein.

In the presence of SKP2 this enables an immune-complex formation involving both labeled antibodies, and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of total protein present in the sample and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Total SKP2 two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Total SKP2 HTRF detection reagents. This protocol allows the cells' viability and confluence to be monitored.

Total SKP2 one-plate assay protocol

Detection of Total SKP2 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol allows miniaturization while maintaining robust HTRF quality.

SKP2 degradation induced by MLN4924 in HeLa cells

HeLa cells were seeded in a 96-well culture-treated plate (40,000 cells/well) in complete culture medium and incubated overnight at 37°C, 5% CO2. The cells were then treated for 18h with increasing concentrations of MLN4924, an inhibitor of Cullin neddylation that promotesSKP2 degradation. After treatment, the cells were lysed with 50 µL of supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking.

For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and supplemented with 4 µL of the HTRF Total SKP2detection reagents. After overnight incubation, the HTRF signal was recorded with an Envision Nexus reader . Additionally, AKT has been described as participating in SKP2 regulation by protecting it from Cdh1-mediated ubiquitination degradation. The activation of AKT under SKP2 inhibition conditions was monitored in the same experiment with the HTRF Total AKT or HTRF Phospho-AKT (Ser473) Detection Kits (Revvity, #64NKTPEG & #64AKSPEG). The corresponding HTRF signals were recorded with an Envision Nexus reader after 4h incubation for Phospho-AKT (Ser473) and overnight incubation for Total AKT.

In parallel, the effect of SKP2 inhibition on p27 (ubiquitinylation target of SKP2) were monitored with the AlphaLISA SureFire Ultra Human and Mouse p27 KIP1 Total Detection Kit (#ALSU-TP27K). 10 µL of cell lysate were transferred into an AlphaPlate-384 microplate (Revvity, #6005350) and 10 µL of the corresponding detection reagents were added. After a 2h incubation, the Alpha signal was recorded with an Envision Nexus reader.

Finally, cell viability was assessed with the ATPlite 1step Kit (Revvity, #6016736/1/9). 5 µL of the same cell lysate were transferred into an HTRF 96-well low volume white plate, and 25 µL of ATPlite 1step detection reagent were added. After a 10-min incubation, the luminescence signal was measured with an Envision Nexus reader.

As expected, the Cullin neddylation inhibitor MLN4924 induced SKP2 degradation and an increased p27 KIP1 level as well as phosphorylation of AKT on Ser473, without affecting the Total AKT signal. In addition, MLN4924 did not induce cytotoxic effects, as measured by the cell viability indicator ATPLite (data not shown).

SKP2 degradation induced by Dioscin in HeLa cells

HeLa cells were seeded in a 96-well culture-treated plate (40,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated for 18h with increasing concentrations of Dioscin, an inhibitor of SKP2 that induces its ubiquitination and its degradation by APC.

After treatment, the cells were lysed with 50 µL of supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total SKP2 detection reagents were added. After overnight incubation, the HTRF signal was recorded with an Envision Nexus reader.

In parallel, the effects of SKP2 inhibition on p27 were monitored using the AlphaLISA SureFire Ultra Human and Mouse p27 KIP1 Total Detection Kit (#ALSU-TP27K).10 µL of cell lysate were transferred into an AlphaPlate-384 microplate (Revvity, #6005350) and 10 µL of the corresponding detection reagents were added. After a 2h incubation,Tte Alpha signal was recorded with an Envision Nexus reader.

Finally, cell viability was assessed alongside the previous assays with the ATPlite 1step Kit (Revvity, #6016736/1/9). 5 µL of the same cell lysate were transferred into an HTRF 96-well low volume white plate, and 25 µL of ATPlite 1step detection reagent. After a 10-min incubation, the luminescence signal was measured with an Envision Nexus reader.

As expected, the SKP2 inhibitor Dioscin induced SKP2 degradation, which increased the p27 KIP1 level. In addition, Dioscin did not induce cytotoxic effects, as measured by the cell viability indicator ATPLite.

SKP2 degradation induced by Dioscin in HCT-116 cells

HCT-116 cells were seeded in a 96-well culture-treated plate (20,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated for 18h with increasing concentrations of Dioscin, an inhibitor of SKP2 that induces its ubiquitination and its degradation by APC. After treatment, the cells were lysed with 50 µL of supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking.

For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total SKP2 detection reagents were added. After an overnight incubation, the HTRF signal was recorded with an Envision Nexus reader.

In parallel, cell viability was assessed with the ATPlite 1step Kit (Revvity, #6016736/1/9). 5 µL of the same cell lysate were transferred into an HTRF 96-well low volume white plate, and 25 µL of ATPlite 1step detection reagent were added. After a 10-min incubation, the luminescence signal was measured with an Envision Nexus reader.

As expected, the SKP2 inhibitor Dioscin induced SKP2 degradation. In addition, Dioscin did not induce cytotoxic effects, as measured by the cell viability indicator ATPLite.

SKP2 expression decrease induced by Bortezomib in HeLa cells

HeLa cells were seeded in a 96-well culture-treated plate (40,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated for 18h with increasing concentrations of Bortezomib, an inhibitor of the 26S proteasome that suppresses the expression of SKP2.

After treatment, the cells were lysed with 50 µL of supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total SKP2 detection reagents were added. After an overnight incubation, the HTRF signal was recorded with an Envision Nexus reader.

In parallel, cell viability was assessed with the ATPlite 1step Kit (Revvity®, #6016736/1/9). 5 µL of the same cell lysate were transferred into an HTRF 96-well low volume white plate, and 25 µL of ATPlite 1step detection reagent were added. After a 10-min incubation, the luminescence signal was measured with an Envision Nexus reader.

As expected, Bortezomib induced an SKP2 signal decrease. In addition, Bortezomib did not induce cytotoxic effects, as measured by the cell viability indicator ATPLite.

Specificity of Total SKP2 assay using siRNA

HeLa cells were seeded in a 96-well culture-treated plate (25,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The next day, the cells were transfected with ON-TARGETplus SMARTPool siRNA SKP2 (Horizon Discovery/Revvity) SKP2, as well as with a non-targeting siRNA used as negative control using DharmaFECT1 transfection reagent. After a 24h-incubation, the medium was replaced by fresh culture medium, and cells were incubated for an additional 24 hours. Following culture medium removal, cells were lysed with 25 µl of supplemented lysis buffer #1 (1X) + BR (1X) for 30 min at room temperature under gentle shaking.

Following cell lysis, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF SKP2 detection antibodies were added. After an overnight incubation, the HTRF signal was recorded with an Envision Nexus reader.

In parallel, cell viability was assessed with the ATPlite 1step Kit (Revvity, #6016736/1/9). 5 µL of the same cell lysate were transferred into an HTRF 96-well low volume white plate, and 25 µL of ATPlite 1step detection reagent were added. After a 10-min incubation, the luminescence signal was measured with an Envision Nexus reader.

The siRNA experiment demonstrated that HTRF detection antibodies specifically measure SKP2. It was found that treatment with the SKP2 siRNA induced a dramatic HTRF signal loss. In addition, siRNA did not induce cytotoxic effects, as measured by the cell viability indicator ATPLite.

Assessment of total SKP2 level in various cell lines

HeLa, A431, HCT-116, and THP-1 cells were seeded in a 96-well culture-treated plate (25,000 cells/well for all cell lines except 20,000 cells/well for HCT-116) in complete culture medium, and incubated overnight at 37°C, 5% CO2. After culture medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking.

For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total SKP2 detection reagents were added. After an overnight incubation, the HTRF signal was recorded with an Envision Nexus reader.

The HTRF Total SKP2 assay efficiently detected Total SKP2 in various cellular models expressing different levels of the protein.

Broad dynamic range of Total SKP2 assay

Various cell densities of HeLa and THP-1 cells were seeded in a 96-well culture-treated plate in complete culture medium, and incubated overnight at 37°C, 5% CO2. After culture medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking.

For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total SKP2 detection reagents were added. After an overnight incubation, the HTRF signal was recorded with an Envision Nexus reader.

The HTRF Total SKP2 assay has demonstrated a broad dynamic range.

HTRF Total SKP2 assay compared to Western Blot

HeLa cells were cultured in flasks and incubated for 48h at 37°C, 5% CO2. After culture medium removal, cells were lysed with 3 mL of supplemented Lysis Buffer #1 for 30 min at room temperature under gentle shaking. Equal amounts of cell lysates (13 µL) were used for a side-by-side comparison of Western Blot and HTRF techniques.

Using the HTRF Total SKP2 assay, 600 cells/well were enough to detect a significant signal, while more than 4,900 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF Total SKP2 assay is 8 times more sensitive than the Western Blot technique.