RAS ELISA 试剂盒

1. Raf-1 RBD Capture Plate : One 96-well strip plate (8 x 12).
2. 5X Assay/Lysis Buffer : One 30 mL bottle of 125 mM HEPES, pH 7.5, 750 mM NaCl, 5% NP-40, 50 mM MgCl2, 5 mM EDTA, 10% Glycerol.
3. Assay Diluent : One 50 mL bottle.
4. 10X Wash Buffer : One 100 mL bottle.
5. Substrate Solution : One 12 mL amber bottle.
6. Stop Solution (Part. No. 310808): One 12 mL bottle. 2

Box 2 (shipped on blue ice packs)

1. Stimulated and non-stimulated cell or tissue lysates
2. Ras activators
3. Protease inhibitors
4. 0.5 M EDTA in water
5. 1 M MgCl2
6. 30 °C incubator or water bath
7. Room temperature shaker
8. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
9. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
10. Multichannel micropipette reservoir
11. Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length)

• Raf1 RBD detects only the active form of Ras
• Convenient 96-well strip-well format

• 1X Assay/Lysis Buffer: Mix the 5X Assay/Lysis Buffer briefly and dilute to 1X in deionized water. Just prior to usage, add protease inhibitors such as 1 mM PMSF, 10 μg/mL leupeptin, and 10 μg/mL aprotinin.
• 1X Wash Buffer: Dilute the 10X Wash Buffer to 1X with deionized water. Stir to homogeneity.
• Anti-pan-Ras Antibody: Immediately before use dilute the Anti-pan-Ras Antibody 1:1000 with Assay Diluent. Do not store diluted solutions.
• Secondary Antibody, HRP Conjugate: Immediately before use dilute the Secondary Antibody, HRP Conjugate 1:2500 with Assay Diluent. Do not store diluted solutions. 3

Note: It is advisable to use fresh cell or tissue lysates because GTP-Ras is quickly hydrolyzed to GDP-Ras, frozen lysates stored at -70 °C may be used. Performing steps at 4 °C or on ice may reduce hydrolysis. Avoid multiple freeze/thaw cycles of lysates.

I. Adherent Cells

1. Culture cells to approximately 80-90 % confluence. Stimulate cells with Ras activator(s) as desired.
2. Aspirate the culture media and wash twice with ice-cold PBS.
3. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer to the cells (0.5 - 1 mL per 100 mm tissue culture plate).
4. Place the culture plates on ice for 10-20 minutes.
5. Detach the cells from the plates by scraping with a cell scraper.
6. Transfer the lysates to appropriate size tubes and place on ice.
7. If nuclear lysis occurs, the cell lysates may become very viscous and difficult to pipette. If this occurs, lysates can be passed through a 271/2-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifugation for 10 minutes (14,000 x g at 4 °C).
9. Collect the supernatant and store samples on ice for immediate use, or snap freeze samples and store at -70 °C for future use.
10. Proceed to GTPγS/GDP Loading for positive and negative controls, or the Activation ELISA (Assay Protocol Section).

II. Suspension Cells

1. Culture cells and stimulate with Ras activator(s) as desired.
2. Perform a cell count, and then pellet the cells by centrifugation.
3. Aspirate the culture media and wash twice with ice-cold PBS.
4. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer to the cell pellet (0.5 - 1 mL per 1 x 107 cells).
5. Lyse the cells by repeated pipetting.
6. Transfer the lysates to appropriate size tubes and place on ice.
7. If nuclear lysis occurs, the cell lysates may become very viscous and difficult to pipette. If this occurs, lysates can be passed through a 271/2-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifugation for 10 minutes (14,000 x g at 4 °C).
9. Collect the supernatant and store samples on ice for immediate use, or snap freeze samples and store at -70 °C for future use. 4
10. Proceed to GTPγS/GDP Loading for positive and negative controls, or the Activation ELISA (Assay Protocol Section).

I. GTPγS/GDP Loading (Positive and Negative Controls) Note: Samples that will not be GTPγS/GDP loaded may be kept on ice during preparation of GTPγS/GDP loading samples.

1. Aliquot 0.5 mL of each cell lysate to two microcentrifuge tubes. Note: Typical protein concentration of sample is > 0.5 mg/mL.
2. Add 10 μL of 0.5 M EDTA to each sample.
3. Add 5 μL of 100X GTPγS to one tube (positive control) and 5 μL of 100X GDP to the other tube (negative control). Mix and label each tube appropriately.
4. Incubate the tubes for 30 minutes at 30 °C with agitation.
5. Stop the loading by adding 33 μL of 1 M MgCl2 to each tube. Mix and place tubes on ice.
6. Continue with the Activation ELISA.

II. Ras Activation ELISA Note: Samples and controls should be thawed/maintained on ice just prior to use (Step 3).

1. Determine the number of wells to be used, and dilute the Raf-1 RBD 1:500 in Assay Diluent. Add 100 μL of the diluted Raf-1 RBD to each well of the Raf-1 RBD Capture Plate. Incubate at room temperature for 1 hour on an orbital shaker. Note: Do not store diluted solutions.
2. Wash microwell strips 3 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.
3. Add 50 μL of Ras lysate sample (10-100 μg), control, or buffer blank per well. Each sample should be assayed in duplicate. Any sample dilutions should be performed in cold, 1X Assay/Lysis Buffer.
4. Immediately add 50 μL of Assay Diluent to each well (100 μL total volume). Incubate at room temperature for 1 hour on an orbital shaker.
5. Wash microwell strips 5 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer. 5
6. Add 100 μL of the diluted Anti-pan-Ras Antibody to each well. Incubate at room temperature for 1 hour on an orbital shaker.
7. Wash the strip wells 5 times according to step 5 above.
8. Add 100 μL of the diluted Secondary Antibody, HRP Conjugate to each well. Incubate at room temperature for 1 hour on an orbital shaker.
9. Wash the strip wells 5 times according to step 5 above. Proceed immediately to the next step.
10. Warm Substrate Solution to room temperature. Add 100 μL of Substrate Solution to each well, including the blank wells. Incubate at room temperature on an orbital shaker. Actual incubation time may vary from 5-20 minutes. Note: Watch plate carefully, if color changes rapidly, the reaction may need to be stopped sooner to prevent saturation.
11. Stop the enzyme reaction by adding 100 μL of Stop Solution into each well, including the blank wells. Results should be read immediately (color will fade over time).
12. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wave length. 6 Example of Results The following figure demonstrates typical results seen with Cell Biolabs 96-well Ras Activation ELISA Kit. One should use the data below for reference only. Figure 1: EGF Stimulation. HeLa cells were serum starved for 18 hours before EGF stimulation (50 ng/mL for 2 minutes). Lysates were then prepared according to Assay Protocol. Background has been subtracted from data. Figure 2: Pan-Ras Antibody Specificity. Anti-pan-Ras Antibody specificity to purified, H-, K-, and N-Ras human isoforms by dot blot.