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KRAS In-well Lysis ELISA Kit Overview
The KRAS In-well Lysis ELISA Kit is designed specifically for high throughput quantification of KRAS activation and inhibition in cell lysates via chemiluminescent detection. By using a concentrated lysis buffer, cells can be lysed directly in 96-well cell culture plates without the requirement for intermediary steps like washing and centrifugation before transfer to the ELISA. Additionally, a highly specific human KRAS antibody is used to detect active KRAS without cross-reactivity with other isoforms. Finally, excess reagents are included to enable high throughput multichannel pipetting using reagent reservoirs and automated microplate washers.
The KRAS In-well ELISA Kit uses the unique binding properties of the RAS binding domain of the c-Raf kinase protein (Raf-RBD) which only binds activated (GTP bound) RAS proteins. A GST-fused, Raf-RBD protein is first bound to the 96-well glutathione-coated ELISA plate. Cells containing activated RAS can be lysed directly in culture wells using a concentrated 5X lysis buffer and then transferred directly to the ELISA plate where only activated RAS binds to the GST-Raf-RBD. Bound activated KRAS is then detected by incubation with a primary antibody that is highly specific for the KRAS isoform. 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.
KRAS In-well Lysis ELISA Kit Highlights
- Fast – Concentrated lysis buffer allows for in-well lysis of cells directly in media, speeding up workflow.
- Isoform Specific – Highly specific recombinant KRAS antibody detects activated human KRAS without cross reactivity from HRAS or NRAS.
- HTP Friendly – Excess reagents are provided so they can be used with reagent reservoirs or automated microplate washers.
- Versatile – Use the concentrated lysis buffer for the in-well lysis workflow or dilute the lysis buffer for use with washed cells. Additional 5X Lysis Binding Buffer is available separately.
- Assay Input – Use with 2 x 104 - 32 x 104 seeded cells per well or with 5-50 µg whole cell extract.
Ras GTPase Background
Small GTPase RAS proteins bind to and hydrolyze GTP, allowing them to function as molecular switches, cycling between an active (GTP-bound) and inactive (GDP-bound) state. This switch regulates signal transduction pathways involved in cellular functions including proliferation, differentiation, and apoptosis. RAS proteins exist as three major isoforms: HRAS, NRAS, and KRAS, each containing about 190 amino acids that share 80-90% sequence identity.
RAS is one of the most frequently mutated genes in cancer. Gain-of-function missense mutations which promote oncogenesis cluster at codons 12, 13, and 61 of KRAS, NRAS and HRAS resulting in enhanced GTP binding due to fast exchange of nucleotide and/or impairment of GAP (GTPase Activating Protein) binding. Although RAS mutations are all activating, they vary in their oncogenic potential and frequency in different tissues, with KRAS mutations being the most prevalent. Because most researchers are looking for isoform specific therapeutics, the KRAS In-well ELISA Kit was designed to be highly specific for the KRAS isoform.
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KRAS In-well Lysis ELISA Kit Contents
Each KRAS 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. 52100 | Cat No. 52105 | Cat No. 52110 | Storage | |
|---|---|---|---|---|
| Reactions | 96 | 5 x 96 | ||
| GST-Raf-RBD | 110 µl | 5 x 110 µl | -80°C | |
| Positive Control Extract (AsPC-1) | 42 µl | 5 x 42 µl | -80°C | |
| Recombinant KRAS Antibody | 15 µl | 44 µl | -20°C | |
| Anti-rabbit HRP-conjugated IgG | 15 µl | 15 µl | -20°C | |
| Protease Inhibitor Cocktail (PIC) | 2 x 105 µl | 1.2 ml | 1.2 ml | -20°C |
| 5X Lysis Binding Buffer | 4 ml | 22 ml | 22 ml | 4°C |
| 10X Wash Buffer AM2 | 2 x 25 ml | 2 x 125 ml | 4°C | |
| 10X Antibody Binding Buffer AM2 | 2.5 ml | 12.5 ml | 4°C | |
| Chemiluminescent Reagent | 2 ml | 10 ml | 4°C | |
| Reaction Buffer | 4 ml | 20 ml | 4°C | |
| 96-well assay plate | 1 ea | 5 ea | 4°C | |
| Plate sealer | 2 ea | 10 ea | 4°C |
KRAS In-well Lysis ELISA Kit Data
Figure 1: The recombinant KRAS antibody used in the KRAS In-well Lysis ELISA Kit is highly specific for human KRAS.
A western blot compares the detection of recombinant human HRAS, KRAS and NRAS isoforms using a Pan-RAS antibody (ProteinTech, Cat. No. 60309-1-Ig) or the Active Motif recombinant KRAS antibody provided in the KRAS In-well Lysis ELISA Kit. Primary antibodies were used at a concentration of 1 µg/ml. 100 ng each of the following recombinant proteins were loaded: human HRAS (2-186, G12V); human KRAS (2-185, G12C/Q61H); and human NRAS (1-186, WT) (Creative Biomart, cat no. HRAS-228H, cat no. KRAS-457H, cat no. NRAS-447H).
Figure 2: Relative Light Units indicate changing levels of active KRAS in AsPC-1 cells using the KRAS In-well Lysis ELISA protocol. AsPC-1 cells are derived from a human pancreatic cancer expressing the KRAS-G12D mutation.
A: AsPC-1 cells were seeded into a 96 well culture plate using a two-fold dilution series starting with 640,000 cells per well and incubated for 2 days. In-well lysis was performed by adding 25 µl 5X Complete Lysis Binding Buffer to each well and incubating on ice for 15 mins. 50 µl cell lysate was then transferred to the Raf-RBS coated ELISA plate. The KRAS In-well Lysis ELISA protocol was followed as written in the manual. The plate was read in the CLARIOstar plate reader (ISOGEN Life Science) using the luminescence setting and a gain setting of 2700.
B: AsPC-1 cells were seeded into a 96 well culture plate using a two-fold dilution series starting with 150,000 cells per well and incubated for 2 days. Cells were then treated with 33 nM MRTX 1133 (a selective KRAS-G12D inhibitor, Cayman Chemical) for three hours. In-well lysis was performed by adding 25 µl 5X Complete Lysis Binding Buffer to each well and incubating on ice for 15 mins. 50 µl cell lysate was then transferred to the Raf-RBD coated ELISA plate. The KRAS In-well Lysis ELISA protocol was followed as written in the manual. The plate was read in the CLARIOstar plate reader (ISOGEN Life Science) using the luminescence setting and a gain setting of 3000.
Figure 3: IC50 of MRTX1133 inhibition of active KRAS-G12D in cell lysate from AsPC-1 using the KRAS In-well Lysis ELISA Kit.
3.2 x 105 AsPC-1 cells were seeded in 96 well plates and grown for 48 hours. Cells were then treated with MRTX1133 (a selective KRAS-G12D inhibitor, Cayman Chemical) at a range of concentrations from 2 - 33 nM for three hours before use in the KRAS In-well Lysis ELISA Kit protocol, with four replicates each. AsPC-1 cells are derived from a human pancreatic cancer expressing the KRAS-G12D mutation.
Figure 4: Increasing active KRAS signal and assay window with increasing amounts of positive control AsPC-1 Cell Lysate
AsPC-1 cells were lysed following the KRAS In-Well Lysis ELISA protocol for preparation of whole-cell extract. Protein was quantified using the Pierce BCA Protein Assay Kit and diluted in 1X Lysis Binding Buffer. 50 µl was added to each well containing the indicated quantity of total protein. RLU is shown on the y-axis while the assay window (signal/background RLU) is indicated above each sample.
Figure 5: Detection of active KRAS in human tumor samples
Human tumors were harvested, and flash frozen from mice that were treated with either a vehicle or 20 mg/kg of KRAS G12C inhibitor AMG-510. 5-10 mg of tumor samples were powderized using a Covaris Cryo Prep system and then mixed with 200 µl 1X Lysis Binding Buffer. Tumor lysates were centrifuged at 15,000 x g for 10 minutes at 4 °C. The supernatant was transferred to a cryotube and stored at -80 °C. A BCA assay was used to determine protein concentration. 25 µg of tumor lysate was added to each well of the KRAS ELISA plate.
KRAS In-well Lysis ELISA Kit FAQs
Can I use frozen cells as starting material?
For best results, we recommend using fresh material as freeze-thawing the starting material may mask experimental effects. If a frozen sample is the only option, we recommend thawing and performing the homogenization step in the Complete Lysis Buffer. Whole cell extracts may be aliquoted and stored at -80°C. Avoid freeze/thaw cycles.
Does the KRAS antibody detect mutant forms of KRAS?
The recombinant KRAS antibody has been tested in the KRAS In-well Lysis ELISA using cell lysates from the AsPC-1 cell line, a pancreatic cancer line expressing the KRAS G12D mutant protein. The recombinant KRAS antibody was also tested and detected the recombinant KRAS G12C, Q61H mutant protein in Western blot. It is expected that it will work with all common active KRAS mutant proteins. However, not all mutations have been tested in the ELISA.
What controls can be used with the kit?
AsPC-1 cell extract is provided as a positive control for KRAS activation in the kit. Sufficient extract is supplied for 4 reactions per 96-well plate. This extract is optimized to give a strong signal (>10x assay window) when used with 10 µl/well.
KRAS In-well Lysis ELISA Kit Documents
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