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AlphaLISA SureFire Biotin-Free Human Total CDK6 Detection Kit, 500 Assay Points

The AlphaLISA™ SureFire® Biotin-Free Human Total CDK6 assay is a sandwich immunoassay for quantitative detection of total CDK6 in cellular lysates using Alpha Technology.

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Feature	Specification
Application	Cell Signaling
Protocol Time	2h at RT
Sample Volume	10 µL

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The AlphaLISA™ SureFire® Biotin-Free Human Total CDK6 assay is a sandwich immunoassay for quantitative detection of total CDK6 in cellular lysates using Alpha Technology.

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ASBF-TCDK6-A-HV

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USD 737.00

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ASBF-TCDK6-A500

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USD 2,490.00

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ASBF-TCDK6-A10K

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USD 14,982.00

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ASBF-TCDK6-A50K

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USD 47,624.00

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For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).

Product information

Overview

Cyclin-Dependent Kinase 6 (CDK6) is a serine/threonine kinase closely related to CDK4 that similarly partners with D-type cyclins to phosphorylate Rb and promote G1/S cell cycle progression. While CDK4 and CDK6 share overlapping functions in Rb phosphorylation, CDK6 has distinct expression patterns and non-redundant biological roles, particularly in hematopoietic cells where it is highly expressed and regulates lymphoid and myeloid differentiation. CDK6 has been shown to function as a transcriptional regulator independently of its kinase activity, directly controlling expression of genes including VEGFA and key hematopoietic transcription factors. CDK6 is frequently overexpressed or amplified in hematologic malignancies including T-cell acute lymphoblastic leukemia, mantle cell lymphoma, and acute myeloid leukemia, as well as in solid tumors. CDK4/6 inhibitors palbociclib, ribociclib, and abemaciclib inhibit both CDK4 and CDK6, and their differential activity against CDK6 may contribute to distinct clinical profiles including differences in neutropenia and efficacy in hematologic cancers. The non-catalytic functions of CDK6 in transcriptional regulation represent potential mechanisms of resistance to CDK4/6 inhibitors that are under active investigation.

The AlphaLISA SureFire Biotin-Free Human Total CDK6 Detection Kit is a sandwich immunoassay for the quantitative detection of total CDK6 in cellular lysates, using Alpha Technology.

Formats:

The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.

AlphaLISA SureFire Biotin Free kits are compatible with:

Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Biotin rich samples

AlphaLISA SureFire Biotin Free kits can be used for:

Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies

How it works

Total-AlphaLISA SureFire Biotin Free assay principle

The Total-AlphaLISA SureFire Biotin Free assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).

The Total-AlphaLISA SureFire Biotin Free assay uses two antibodies which recognize two different distal epitopes on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with a proprietary CaptSure3 agent to capture one of the detection antibodies, which is labeled with the CaptSure3 tag. In the presence of target protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.

Total-AlphaLISA SureFire Ultra Biotin Free assay principle

Total-AlphaLISA SureFire Biotin Free two-plate assay protocol

The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Total-AlphaLISA SureFire Biotin Free detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

2 plates assay protocol alphalisa surefire ultra biotin free total assay

Total-AlphaLISA SureFire Biotin Free one-plate assay protocol

Detection of Total target protein with AlphaLISA SureFire Biotin Free reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire quality.

1 plate assay protocol alphalisa surefire ultra biotin free total assay

Assay validation

Induction of CDK6 expression in MCF7 cells treated with Neuregulin-1

MCF7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were treated with 5 µM LY294002 (PI3K inhibitor) for 3 hours then increasing concentrations of Neuregulin-1 (NRG-1) for 24 hours in serum free media.

After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CDK6 and ERK Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 8,000 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, NRG-1 treatment resulted in a modest increase of CDK6 levels.

Pharmacological Validation (Inhibition) of CDK6 Total assay

Degradation of CDK6 in cells treated with CDK6 PROTAC

MOLT-4 cells were seeded in a 96-well plate (50,000 cells/well) in complete RPMI 1640 and treated with increasing concentrations of BSJ-03-123 for 24 hours at 37°C, 5% CO₂.

After treatment, the cells were spun down and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Cell lysates were further diluted in Lysis Buffer. CDK6 Total levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 5,000 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, treatment with BSJ-03-123 resulted in a dose-dependent decrease of CDK6 levels while ERK levels remained unchanged (data not shown).

Degradation of Total CDK6 in cells treated with CDK4/6 PROTAC

MCF7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂.Cells were treated with increasing concentrations of BSJ-03-204 for 24 hours.

After treatment, the cells were lysed with 50 µL or 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Total CDK6 and Total ERK levels were evaluated using AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 8,000 cells for CDK6 or 2,000 cells for ERK) 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, treatment with BSJ-03-204, a CDK4/6 PROTAC, resulted in a dose-dependent decrease of CDK6 while ERK levels remained unchanged.

Assay sensitivity

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from MOLT-4 cells lysed with 5X Lysis Buffer at 2 x 10⁶ cells/mL for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and CDK6 levels were evaluated by AlphaLISA SureFire Biotin Free. 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. This assay can detect CDK6 expression in less than 200 cells/datapoint.

Specifications

Other specifications

Application	Cell Signaling
Automation Compatible	Yes
Brand	AlphaLISA SureFire Biotin-Free
Detection Modality	Alpha
Product Group	Kit
Protocol Time	2h at RT
Sample Volume	10 µL
Shipping Conditions	Shipped in Blue Ice
Target	CDK6
Target Class	Phosphoproteins
Target Species	Human
Technology	Alpha
Therapeutic Area	Oncology
Unit Size	500 Assay Points