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AlphaLISA SureFire Ultra Human & Mouse Phospho-RAF1 (Ser259) Detection Kit, 100 Assay Points

The AlphaLISA™ SureFire® Ultra™ Human and Mouse Phospho-Raf-1 (Ser259) assay is a sandwich immunoassay for quantitative detection of phospho-Raf-1 in cellular lysates using Alpha technology.

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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Feature Specification
Application Cell Signaling
Sample Volume 30 µL

The AlphaLISA™ SureFire® Ultra™ Human and Mouse Phospho-Raf-1 (Ser259) assay is a sandwich immunoassay for quantitative detection of phospho-Raf-1 in cellular lysates using Alpha technology.

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).

Click to copy promo code to clipboard.
SAVE 15% NOW on online orders. Use promo code below.
YEAREND15
Offer valid until 12/15. Terms and conditions apply.
Product Variants
Unit Size: 100 Assay Points
Part #:
ALSU-PRAF-B-HV
List Price
USD 694.00
Unit Size: 500 Assay Points
Part #:
ALSU-PRAF-B500
List Price
USD 2,346.00
Unit Size: 10,000 Assay Points
Part #:
ALSU-PRAF-B10K
List Price
USD 14,270.00
Unit Size: 50,000 Assay Points
Part #:
ALSU-PRAF-B50K
List Price
USD 46,060.00

Overview

RAF proto-oncogene serine/threonine-protein kinase has numerous aliases, including Raf-1, c-Raf, and proto-oncogene c-RAF. Raf-1 is an enzyme belonging to the subfamily of membrane associated GTPases. Raf-1 is a principal component of the mitogen-activated protein kinase (MAPK) pathway, specifically the ERK1/2 signaling cascade 1. Raf-1 regulates cell growth and division, playing a key role in cell proliferation.  It is also involved in cell survival pathways and metabolic processes within cells. This protein is associated with various cancers, including bladder urothelial carcinoma, skin cutaneous melanoma, and uterine corpus endometrial carcinoma.

The AlphaLISA SureFire Ultra Human and Mouse Phospho-Raf-1 (Ser259) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-Raf-1 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 Ultra kits are compatible with:

  • Cell and tissue lysates
  • Antibody modulators
  • Biotherapeutic antibodies

Alpha SureFire kits can be used for:

  • Cellular kinase assays
  • Receptor activation studies
  • High-throughput drug screening

Specifications

Application
Cell Signaling
Automation Compatible
Yes
Brand
AlphaLISA SureFire Ultra
Detection Modality
Alpha
Lysis Buffer Compatibility
Lysis Buffer
Molecular Modification
Phosphorylation
Product Group
Kit
Sample Volume
30 µL
Shipping Conditions
Shipped in Blue Ice
Target
Raf-1
Target Class
Phosphoproteins
Target Species
Human
Mouse
Technology
Alpha
Therapeutic Area
Oncology
Unit Size
100 Assay Points

Assay validation

Inhibition of Phospho Raf-1 (Ser259)

HeLa cells were seeded in a 96-well plate(40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated for 24 hours with increasing concentrations of Geldanamycin prepared in serum free medium containing 1% FBS.

After treatment, the cells were lysed with 100µL of lysis buffer for 10 minutes at RT with shaking (350 rpm). Raf-1 Phospho (Ser259) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate(approximately 4,000 cells) were transferred into a 384-well white OptiPlate, followed by 5µL 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 Nexus™ using standard AlphaLISA settings.

As expected, Geldanamycin (HSP90 inhibitor, disrupts the Raf-1-HSP90 complex) triggered a dose-dependent decrease in the levels of Raf-1 Phospho (Ser259) and Total.

Inhibition of Phospho Raf-1 (Ser259)

Assay specificity/selectivity

Selectivity of Phospho Raf-1 (Ser259) assay

Raf-1 or Raf-1 p-S259 peptides were titrated into a fixed dilution of positive control lysate (HeLa cells treated with 200 ng/mL PMA). RAF1 Phospho (Ser259) levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of peptide mix 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 Nexus using standard AlphaLISA settings.

Raf-1 p-Ser259 peptide competition

Phospho Raf-1 (Ser259) levels were assessed in HeLa cells (WT) and Raf-1 knockout (KO) HeLa cells. Raf-1 KO cells (Abcam ab264978) and WT-HeLa cells were cultured to confluency in T25 flasks in complete medium at 37°C, 5% CO2.

Each flask was lysed in 2 mL of lysis buffer for 10 minutes at RT with shaking (350 rpm). Phospho Raf-1 (Ser259) levels were then evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate 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 Nexus using standard AlphaLISA settings. As expected, Phospho Raf-1 (Ser259) was detected in the WT-Hela cells but not in the Raf-1 KO cell line.

Raf-1 p-Ser259 Knockout Validation

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