| SKU-Pack Size | Availability | Price | Quantity |
| abs955-50tests | In stock | $420.00 | - + |
Request bulk quotation| The name of the reagent | volume | Preservation conditions |
| Lysis buffer | 30ml | 4 ℃ |
| 10*Wash buffer | 50ml | 4 ℃ |
| 1*SDS Sample buffer | 1ml | 4 ℃ |
| Protein A Agarose beads | 250ul | 4 ℃ |
| Protein GAgarose beads | 250ul | 4 ℃ |
2.use method
A. Prepare cell lysates
1. Blot the medium dry. A fresh medium containing regulatory molecules was added to allow the cells to be treated within a predetermined time.
2. Cells were collected and washed once with 1 X PBS precooled with ice after removing the culture medium.
3. Remove PBS and add 0.5 mL ice precooled Lysis buffer to each cell plate (10 cm) and incubate on ice for at least 5 minutes.
4. Scrape the cells from the plate and transfer the extract to a microcentrifuge tube. On ice.
5. Perform ultrasonic crushing on the ice for 3 times, 5 seconds each time.
6. Microcentrifugation at 4 °C at 14,000 x g for 10 min. Transfer supernatant to new tube. The supernatant is the cell lysate. If necessary, the pyrolysis may be stored at -80 °C.
Note: After the cell lysates are prepared, western blotting can be performed to determine the presence of the target protein in the cell lysates.
B. Immunoprecipitation method
A) Cell lysate pre-clearance (optional step)
1. Add 5 μ L Protein A and 5 μ L Protein G to 500 μ L (containing 200-1000 ug total Protein) cell lysate.
2. Rotate and incubate at 4 °C for 30-60 minutes.
3. Centrifugation at 12000g at 4°C for 1 min. Reserve supernatant.
B) antigen antibody binding
1. Add 500 μ L (200 -- 1000 ug total protein) cell lysate (can be pre-cleaned cell lysate) to the new centrifuge tube.
2. Add monoclonal/polyclonal antibody (1-5 μg) to target protein.
3. Homologous antibodies of non-specific immunity were used as control.
4. Blend gently at 4 °C overnight.
C) Precipitation of immune complexes
1. After incubation overnight, add 5 μ L Protein A and 5 μ L Protein G.
2. Mix gently at 4 °C for 1-3 hours or overnight.
3. Centrifuge at 12,000 g for 1 minute, retain the precipitation.
4. Use 0.5 ml 1*Wash buffer to clean the precipitation, centrifuge at 12,000 g for 1 minute, and retain the precipitation.
5. Repeat step 4 for a total of 3 times.
Note: When cleaning and removing the supernatant, care should be taken to avoid suction of the filler
C. Western blotting was used for analysis
1. Suspend the precipitate in 20-40 μ L 1*SDS sample buffer, swirl the precipitate, then centrifuge for 30 seconds to remove beads and liquid from tube wall to tube bottom.
2. Heat the sample to 95-100 °C for 2-5 minutes, then centrifuge at 14,000 g for 1 minute, remove supernatant.
3. Load sample (15 -- 30 μ L) onto SDS-PAGE gel.
4. Samples were analyzed by western blotting.
3. Method of kit preservation
The kit is stored at 4℃ and effective for 1 year.
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