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AlphaLISA SureFire Biotin-Free Human Total HBS1L Detection Kit, 50,000 Assay Points

The AlphaLISA™ SureFire® Biotin-Free Human Total HBS1L assay is a sandwich immunoassay for quantitative detection of total HBS1L in cellular lysates using Alpha Technology.

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Feature	Specification
Application	細胞シグナル伝達
Protocol Time	2h at RT
Sample Volume	10 µL

The AlphaLISA™ SureFire® Biotin-Free Human Total HBS1L assay is a sandwich immunoassay for quantitative detection of total HBS1L in cellular lysates using Alpha Technology.

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Unit Size: 100 Assay Points

Part #:

ASBF-THBS1L-A-HV

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Unit Size: 500 Assay Points

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ASBF-THBS1L-A500

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Unit Size: 10,000 Assay Points

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ASBF-THBS1L-A10K

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Unit Size: 50,000 Assay Points

Part #:

ASBF-THBS1L-A50K

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).

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Product information

Overview

HBS1L (HBS1-Like Translational GTPase) is a member of the HBS1-like family of translational GTPases related to eukaryotic release factor 3 (eRF3) that functions in ribosome quality control and mRNA surveillance pathways. HBS1L participates in the No-Go Decay (NGD) and Non-Stop Decay (NSD) pathways, which resolve stalled ribosomes on problematic mRNA transcripts containing strong secondary structures, rare codons, or absent stop codons. In these pathways, HBS1L cooperates with the endonuclease PELO (Pelota) to recognize and split stalled ribosomal complexes, initiating downstream mRNA degradation and recycling of ribosomal subunits. Beyond its role in mRNA quality control, HBS1L has been identified as a regulator of fetal hemoglobin (HbF) expression through genetic studies of the HBS1L-MYB intergenic region on chromosome 6q23, where common variants influence HbF levels and modify the severity of sickle cell disease and β-thalassemia. HBS1L expression levels and variants in the HBS1L-MYB locus are among the strongest known genetic modifiers of hemoglobin switching, positioning HBS1L as a relevant target in hemoglobinopathy research.

The AlphaLISA SureFire Biotin-Free Human Total HBS1L Detection Kit is a sandwich immunoassay for the quantitative detection of total HBS1L 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

Time dependent degradation of HBS1L

MOLT-4 cells were seeded in a 96 well plate (200,000 cells/well) in complete medium and incubated with 2.5 µM CC-885 for increasing length of time 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). HBS1L levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 20,000 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.

CC-885 is a cereblon E3 ligase modulator that induces the degradation of HBS1L, levels decrease with treatment over time.

Molecular glue substrate selectivity

MOLT-4 cells were seeded in a 96 well plate (100,000 cells/well) in complete medium and incubated with increasing concentrations of CC-885 or CC-90009 for 8 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). HBS1L and GSPT1 levels were evaluated using respective AlphaLISA SureFireassays. For the detection step, 10 µL of cell lysate (approximately 10,000 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.

CC-885 and CC-90009 are both molecular glues that induce the degradation of GSPT1. CC-885 has a broad selectivity and degrades GSPT1 and HBS1L. CC-90009 is a more selective degrader and does not degrade HBS1L.

Assay validation – molecular glue target selectivity

CC-885 induced degradation of HBS1L in MIA PaCa-2 and A549 cells

MIA PaCa-2 and A549 cells were seeded in a 96 well plate (40,000 cells/well) in complete medium and incubated for 24 hours at 37°C, 5% CO₂.Cells were treated with increasing concentrations of CC-885 for 24 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). HBS1L and ERK levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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.

CC-885 treatment results in degradation of HBS1L in MIA PaCa-2 and A549 cells, a small decrease in ERK Total levels were also observed.

Assay Validation – CC-885 induced degradation

Assay versatility

HBS1L expression in various cell lines

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

HBS1L levels were evaluated using the AlphaLISA SureFire Ultra Biotin Free assay. For the detection step, 10 µL of cell lysate (approximately 4,000 adherent cells or 40,000 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.

HBS1L expression was detected in a range of human cell lines.

Assay sensitivity

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from MIA PaCa-2 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 HBS1L 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 HBS1L expression in less than 100 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	HBS1L
Target Class	Phosphoproteins
Target Species	Human
Technology	Alpha
Therapeutic Area	Oncology
Unit Size	50,000 Assay Points