| Product Description | Product content：
| O665490 | Component | 50 T | Storage | | O665490A | Blocking Buffer | 500 mL | 2-8℃. Do not freeze. | | O665490B | Antibody Pretreat Solution (HRP/Rabbit) | 5×1 mL | 2-8℃. Do not freeze.
| | O665490C | Dilution Buffer | 500 mL | 2-8℃. Do not freeze.
| | O665490D | Wash Buffer (10×) | 500 mL | 2-8℃. Do not freeze. |
|
Product Introduction：
The one-step rapid WB assay kit (rabbit) is the latest Western Blot detection kit developed by Kangwei Century, which can
Obtain high-quality Western Blot results within about 1 hour, with simple operation, high detection sensitivity, low background, and no
Additional secondary antibodies need to be added, with strong system stability. The conventional Western Blot indirect detection process (blocking, primary antibody binding)
Combining with secondary antibodies requires a long time, a complex experimental process, and requires multi-step optimization of conditions. The protein on the glue is transferred to
After coating the carrier membrane, incubate it with the blocking solution in the reagent kit for 5 minutes, and then incubate the carrier with the primary antibody treated with antibody reaction solution
After washing the membrane three times (5 minutes each time), it can undergo luminescence or color detection. This reagent kit is designed for target protein one
The use of an experimental system derived from rabbits. Notes:
1. Customers need to prepare their own rabbit source primary antibody.
2. Before using Blocking Buffer blocking solution, Antibody Pretreat Solution (HRP/Rabbit) antibody reaction solution (rabbit), and Wash Buffer (10 x) rinse solution, please mix thoroughly.
3. If there is precipitation in the rinsing solution when stored at 2-8 ℃, please restore it to room temperature, dissolve the precipitation, and use it normally. The 1x rinsing solution can be stored at room temperature for one month.
4. It is recommended to stain the membrane with reagents such as spring red after the transfer is completed, and cut off any excess parts on the membrane to increase the efficiency of the reagents.
5. The optimal dilution amount for primary antibody and antibody reaction solution HRP (rabbit) needs to be determined through preliminary experiments.
6. Antibody reaction solution HRP (rabbit), antibody dilution solution, and antibody dosage can be increased or decreased proportionally according to the size of the membrane.
7. The antibody dilution solution containing the first antibody can be recycled and reused once. Antibodies with low specificity and affinity are not recommended for repeated use. If the recovered antibody is used within 1-2 days and stored at 2-8 ℃ for long-term storage, please freeze it at -20 ℃ to avoid repeated freeze-thaw cycles.
8. If there is a high background, please adjust the amount of antibodies and increase the number of times the film is washed.
9. All reagents in the reagent kit should be stored at 2-8 ℃ to avoid freezing and thawing.
Operation steps:
This product is suitable for the sealing and antibody incubation steps after membrane transfer, taking a 5 cm x 8 cm membrane as an example:
1. Preparation of rinsing solution: Dilute 10 ml of Wash Buffer (10 x) with distilled water to 100 ml, which is 1 x Wash Buffer. Set aside. Use 8-10 ml for each film wash.
2. Sealing: After the membrane transfer is completed, immerse the membrane in 10 ml Blocking Buffer and seal at room temperature for 5 minutes.
3. Rinse: Pour off the sealing solution, add 8-10 ml of 1 x Wash Buffer, and rinse at a high speed on a shaker for 1 minute.
4. Prepare antibody incubation solution while washing the membrane: Take Antibody Pretreat Solution (HRP/Rabbit) 100 μ Add rabbit derived primary antibody 3-10 into the centrifuge tube μ g. Suck and beat the gun head until thoroughly mixed, and incubate at room temperature for 5 minutes. Add to 10 ml Dilution Buffer and mix well. Note: 1) The dosage of primary antibody can also be adjusted according to the dilution of the antibody. Taking the final dilution of antibodies at 1:1000 as an example, take 100 μ Add HRP (rabbit) antibody reaction solution into the EP tube and add 10 μ Add the first antibody to 10 ml of antibody diluent, mix well, and incubate at room temperature for 5 minutes. 2) If the membrane area is small, the amount of antibodies, reaction solution, and diluent can be reduced proportionally.
5. After completing step 3, pour out the rinsing solution and add the antibody incubation solution mixed with primary antibody, Antibody Pretreat Solution (HRP/Rabbit), and Dilution Buffer to the membrane (ensuring that the incubation solution completely submerges the membrane surface). Incubate at room temperature on a shaker at a speed of about 60 rpm for 40 minutes.
6. Discard (recover) the antibody incubation solution and rinse 3-5 times with the prepared 1 x Wash Buffer, each time for 3 minutes.
7. Conduct subsequent testing. It is recommended to use ECL or DAB methods for testing.
Example 1: Antigen 293T cell lysate
A: Ordinary WB control: beta actin rabbit antibody (CW0097) 3.3ug incubated at room temperature for 40 minutes, washed with membrane, secondary antibody sheep anti rabbit HRP (CW0103) diluted at 1:10000, room temperature for 40 minutes, ECL (CW0049) exposed 
Example 2 Antigen is 293T cell lysate
C: Ordinary WB control: PAK1, Epitomics rabbit monoclonal antibody 1:1000, incubated at room temperature for 40 minutes, washed with membrane, secondary antibody sheep anti rabbit HRP (CW0103) diluted at 1:10000, room temperature for 40 minutes, ECL (CW0049) exposed
D: One step WB: Epitomics rabbit monoclonal antibody was incubated at 1:1000 room temperature for 40 minutes, and ECL (CW0049) was exposed.
|
|---|
