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NRAS In-well Lysis ELISA Kit

Fast, sensitive, and highly specific quantification of human NRAS

 

NRAS In-well Lysis ELISA Kit Overview

NRAS In-well Lysis ELISA Kit
 

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The NRAS In-well Lysis ELISA Kit is designed for high throughput quantification of active NRAS in cell lysates via chemiluminescent detection. A GST-fused, Raf-RBD protein, which binds to activated RAS, is first bound to a 96-well glutathione-coated ELISA plate. Cells containing NRAS protein are then lysed in a 96-well cell culture plate before transferring to the Raf-RBD coated ELISA plate. A highly specific human NRAS antibody is then added to detect active NRAS. Addition of a secondary antibody conjugated to horseradish peroxidase (HRP), followed by addition of developing solution provides a sensitive chemiluminescent readout that is easily quantified by luminescence.

NRAS In-well Lysis ELISA Kit Highlights

  • Isoform Specific – Highly specific recombinant NRAS antibody detects activated human NRAS without cross reactivity from HRAS or KRAS.
  • Fast & HTP Friendly – Excess reagents are provided so they can be used with reagent reservoirs or automated microplate washers.
  • Assay Input – Use with 2 x 105 - 5 x 105 seeded cells per well or with 20-50 µg of whole cell extract.
Flow Chart of NRAS In-well Lysis ELISA Kit
Flow Chart of NRAS In-well Lysis ELISA Kit

(Click image to enlarge)

 

NRAS In-well Lysis ELISA Kit Contents

Each NRAS In-well Lysis ELISA Kit is shipped on dry ice and contains reagents requiring multiple storage temperatures. Please confirm receipt of all reagents upon arrival and store items at the appropriate temperatures as indicated below. The kit is guaranteed for 6 months when all components are stored properly.

  Cat No. 52099 Cat No. 52110 Storage
Reactions 96    
GST-Raf-RBD 100 µl   -80°C
Positive Control Extract (SK-MEL-2) 40 µl   -80°C
Recombinant NRAS Antibody 20 µl   -80°C
HRP-conjugated Secondary Antibody 20 µl   -20°C
Protease Inhibitor Cocktail (PIC) 2 x 101 µl 1.2 ml -20°C
5X Lysis Binding Buffer 4 ml 22 ml 4°C
10X Wash Buffer AM2 29 ml   4°C
1X Antibody Binding Buffer 14 ml   4°C
Chemiluminescent Reagent 1.9 ml   4°C
Reaction Buffer 3.8 ml   4°C
96-well assay plate 1 ea   4°C
Plate sealer 4 ea   4°C


 

NRAS In-well Lysis ELISA Kit Data

KRAS western blot

Figure 1: The recombinant NRAS antibody used in the NRAS In-well Lysis ELISA Kit is specific for human NRAS.
Active motif compared the detection of recombinant human HRAS, KRAS, and NRAS isoforms using the recombinant NRAS antibody provided in the NRAS In-well Lysis ELISA Kit. The dot blot (Figure 1A) was spotted with three types of recombinant proteins: human HRAS, human KRAS, and human NRAS. NRAS primary antibody was used at a concentration of 1 µg/ml. The NRAS antibody demonstrated preferential detection of recombinant KRAS over HRAS and NRAS. In 1B, the bead-based ELISA assay was used to confirm the specificity of NRAS antibody. HRAS, KRAS, and NRAS peptides were conjugated to MagPlex Luminex beads and incubated with 200ng/mL, 667, 222, 74, and 25 ng/mL of NRAS antibody. Peptide-bound antibody was detected with anti-mouse IgG-Phycoerythrin and read in a Luminex instrument. The NRAS antibody only detected the NRAS peptide, not HRAS and KRAS. PanRAS antibody detected all three peptides.

KRAS Data Figure 2

Figure 2: Performance of the NRAS In-well Lysis ELISA Kit using SK-MEL2 lysates and cells.
A: Signal detection using SK-MEL-2 cell lysates: SK-MEL-2 whole cell lysates were tested at increasing amounts (0, 20, and 40 μg), resulting in a dose-dependent increase in RLU (purple bars). The assay window (green line) also increased with higher input lysate. Error bars represent standard deviations (n = 3).
B: Signal detection using SK-MEL-2 cells in in-well lysis format: SK-MEL-2 cells were seeded directly into the well at 0 or 500,000 cells/well and lysed in place. High luminescence signal and assay window were observed at 500,000 cells/well, demonstrating compatibility of the assay with in-well lysis. Error bars represent standard deviations (n = 3).

 

 

NRAS In-well Lysis ELISA Kit Documents

 


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