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AlphaLISA SureFire Biotin-Free Human Total PELO Detection Kit, 10,000 Assay Points
AlphaLISA SureFire Biotin-Free Human Total PELO Detection Kit, 10,000 Assay Points
AlphaLISA SureFire Biotin Free Total Kit Schematic
The AlphaLISA™ SureFire® Biotin-Free Human Total PELO assay is a sandwich immunoassay for quantitative detection of total PELO in cellular lysates using Alpha Technology.
| Feature | Specification |
|---|---|
| Application | Cell Signaling |
| Protocol Time | 2h at RT |
| Sample Volume | 10 µL |
The AlphaLISA™ SureFire® Biotin-Free Human Total PELO assay is a sandwich immunoassay for quantitative detection of total PELO in cellular lysates using Alpha Technology.
Product variants
Unit Size: 100 Assay Points
Part #:
ASBF-TPELO-A-HV
List price
USD 737.00
Your price:
Unit Size: 500 Assay Points
Part #:
ASBF-TPELO-A500
List price
USD 2,490.00
Your price:
Unit Size: 10,000 Assay Points
Part #:
ASBF-TPELO-A10K
List price
USD 14,982.00
Your price:
Unit Size: 50,000 Assay Points
Part #:
ASBF-TPELO-A50K
List price
USD 47,624.00
Your price:
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).
AlphaLISA SureFire Biotin-Free Human Total PELO Detection Kit, 10,000 Assay Points
AlphaLISA SureFire Biotin Free Total Kit Schematic
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Product information
Overview
Pelota (PELO) is a highly conserved eukaryotic protein and ortholog of bacterial ArfA that functions as a central component of the ribosome-associated quality control (RQC) and No-Go Decay (NGD) pathways. PELO recognizes stalled ribosomes on aberrant mRNA transcripts — including those lacking stop codons, containing strong secondary structures, or encoding rare codon stretches — and in cooperation with its GTPase partner HBS1L, facilitates endonucleolytic cleavage of the mRNA within the ribosomal A-site. Following mRNA cleavage, PELO and HBS1L promote ribosome splitting by the ABCE1 ATPase, enabling recycling of ribosomal subunits and initiating degradation of the truncated nascent polypeptide by the RQC complex. PELO is essential for maintaining translational fidelity and cellular proteostasis, and its loss leads to ribosome collision accumulation, activation of the integrated stress response, and impaired cell viability. In mammals, PELO plays important roles in spermatogenesis and germ cell development, and conditional knockout studies have revealed requirements in hematopoiesis and stem cell maintenance. PELO has also been identified as a component of the stem cell self-renewal machinery in Drosophila, suggesting conserved roles in stem cell biology across species.
The AlphaLISA SureFire Biotin-Free Human Total PELO Detection Kit is a sandwich immunoassay for the quantitative detection of total PELO 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 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.
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.
Assay validation
Time dependent degradation of PELO
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). PELO 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 induced the degradation of PELO.
MOLT-4 cells treated with CC-885
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 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). PELO and ERK Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 2,500 cells (PELO) or 10,000 cells (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.
CC-885 treatment results in decreased levels of PELO in MOLT-4 cells, a small decrease in ERK Total levels was also observed.
Assay versatility
PELO 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 lysed with Lysis Buffer at a density of 4 x 106 cells/mL.
PELO levels were evaluated using the AlphaLISA SureFire Ultra Biotin Free assay. For the detection step, 10 µL of cell lysate (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.
PELO 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 100 mL of Lysis Buffer for 10 minutes at RT with shaking.
Lysate was serially diluted and PELO 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 PELO expression in less than 50 cells/datapoint.
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 |
PELO
|
| Target Class |
Phosphoproteins
|
| Target Species |
Human
|
| Technology |
Alpha
|
| Therapeutic Area |
Oncology
|
| Unit Size |
10,000 Assay Points
|
Resources
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Guide
AlphaLISA SureFire Ultra: the ultimate guide for successful experiments
The definitive guide for setting up a successful AlphaLISA SureFire Ultra assay
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