AlphaLISA SureFire Biotin-Free Human and Mouse Total LDLR Detection Kit, 100 Assay Points

LDLR accumulation in cells after Bafilomycin A1 treatment

HepG2 cells were seeded in a 96-well plate (50,000 cells/well) in complete medium and incubated for 48 hours at 37°C, 5% CO₂. Cells were treated with increasing concentrations of Bafilomycin A1 for 16 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). LDLR and ERK Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 500 (LDLR) or 5,000 (ERK) cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, Bafilomycin A1 (an inhibitor of lysosomal degradation) treatment resulted in an increase in cellular levels of LDLR while ERK Total levels remained unchanged.

HepG2 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated for 24 hours at 37°C, 5% CO₂. Cells were treated with 5 µM Mevastatin for 24 hours.

After treatment, the supernatant was harvested and cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). LDLR levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 400 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, Mevastatin treatment upregulates the expression of LDLR.

HepG2 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated for 24 hours at 37°C, 5% CO₂. Cells were treated with 25 µM PCSK9-IN-23 for 24 hours.

After treatment, the supernatant was harvested and cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). LDLR levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 400 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

PCSK9-IN-23 is a small molecule inhibitor of PCSK9 and blocks secretion from cells. This reduces PCSK9 binding to cell surface LDLR, resulting in increased LDLR levels.

PCSK9 mediated regulation of LDLR expression in HepG2 cells

HepG2 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated for 24 hours at 37°C, 5% CO₂. Increasing concentrations of recombinant PCSK9, with or without Bafilomycin A1 (200 µM) or Evolucamab (70 µg/mL), were spiked into the cell supernatant and incubated for 4 hours.

After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). LDLR Total levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of diluted cell lysate (approximately 400 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, PCSK9 spiked into the cell supernatant results in the degradation of LDLR. This was prevented by Evolucamab (by blocking PCSK9 mediated LDLR degradation) and Bafilomycin A1 (by inhibiting lysosomal degradation).

Assay specificity - knockout validation

LDLR levels were assessed in HeLa wild type (WT) and LDLR KO (Abcam, ab273838) cell lines cultured to confluence in T175 flasks at 37°C, 5% CO₂.

Each flask was lysed in Lysis Buffer for 10 minutes at RT with shaking and LDLR levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of lysate (approximately 2,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

LDLR was detected in WT but not in KO cells, confirming the specificity of the assay for the detection of LDLR protein.

LDLR expression in various cell lines

Adherent cells were grown to confluence in a T175 flask at 37°C, 5% CO2, and were lysed with Lysis Buffer at a density of 0.4 x 10⁶ cells/mL. Suspension cells were lysed with Lysis Buffer at a density of 4 x 10⁶ cells/mL.

LDLR levels were evaluated using the AlphaLISA SureFire Ultra Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 400 adherent cells or 4,000 suspension cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor Mix and incubated for 1 hour at RT. Finally, 5 µL of Donor Mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

LDLR expression was detected in a range of human and mouse cell lines.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from HepG2 cells cultured to confluence a T175 flask and lysed with 10 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and LDLR levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

Approximate number of cells per datapoint is indicated. The dotted line represents assay background. The assay can detect LDLR expression in less than 50 cells/datapoint.