U-Blot® PVDF imprinting membrane (0.2 μ m)

U-Blot ® PVDF imprinting membrane (0.2 μ m)It is a polyvinylidene fluoride (PVDF) microporous membrane, which is used to transfer proteins from various gel matrices. Compared with nitrocellulose membrane, it has better operational performance.

This hydrophobic membrane has two pore sizes: 0.45 μ m and 0.2 μ m, allowing it to bind proteins of various molecular weight ranges:

• 0.45 μ m aperture: suitable for most proteins, providing higher detection sensitivity.

• 0.2 μ m pore size: suitable for proteins with a molecular weight less than 20000, ensuring better adsorption and retention of small molecule proteins.

U-Blot ® PVDF imprinting membrane has excellent protein retention, high physical strength, and wide chemical compatibility, making it the choice for immunoassays.

U-Blot ® PVDF imprinting membrane (0.2 μ m)

1. Use SDS PAGE gel to separate protein samples and protein markers.

2. Carefully cut and place gel, and balance gel in cold transfer buffer for 5~20 minutes.

3. Cut the membrane and filter paper to the size suitable for gel. Pre wet the PVDF membrane in methanol for 20 seconds until it changes from opaque white to uniform semi transparent gray, then soak it in ultrapure water for 1-2 minutes to remove any residual reagents; Then equilibrate in cold transfer buffer for 2-3 minutes until it can be used.

Usage reminder:

After wetting the film, do not let it dry. If the membrane part becomes dry (opaque white), it must also be rewetted.

4. Assemble the transfer device in turn, transfer clip sponge pad filter paper pre wetted film cut and pre wetted gel filter paper sponge pad transfer clip, and pay attention to avoid wrinkles, folds or bubbles in each layer (use tools such as cutting rubber plate to gently press bubbles to make each layer closely fit)

5. Set the operating conditions according to the instructions of the transfer device used, and start the transfer process.

6. After the transfer, take out the imprint film and wash it in ultrapure water briefly to remove gel debris. Imprinted membranes can be stored by air drying or immediately used for immunoassay steps.

Some key factors for successful Western blot experiments, such as protein concentration, blocking solution, and antibody concentration, may need to be optimized in the following schemes.

Standard immune testing

If the imprinting film has dried, soak it again in methanol for 15 seconds or until its color changes from opaque white to semi transparent gray.

2. Rinse the imprinting membrane with ultrapure water for 1 minute.

3. Place the imprinting membrane in the blocking buffer and gently stir for incubation for 1 hour.

4. Dilute the primary antibody in commercial antibody diluent, washing buffer, or blocking buffer.

5. Place the imprinting membrane in a diluted primary antibody solution and incubate at room temperature for 1 hour (or overnight at 4 ° C), gently stirring.

6. Use washing buffer (containing Tween) ®- Wash the imprinting membrane 3-5 times with a buffer of 20 surfactants (TBST or PBST), each time for 5 minutes.

7. Dilute the secondary antibody in commercial antibody diluent, washing buffer, or blocking buffer.

8. Place the imprinting membrane in a diluted enzyme labeled secondary antibody solution and incubate at room temperature for 1 hour.

9. Wash the imprinting membrane 3-5 times with washing buffer, each time for 5 minutes.

10. Place the imprinting membrane into a clean container and add the corresponding detection reagent.

11. Follow the instructions for the reagents used in the test and incubate for 1-5 minutes.

(1) For HRP or AP chemiluminescence reagents, expose the imprinting membrane to X-ray film or use an imaging system to obtain digital images.

(2) For colorimetric detection, add reagents and wait for the signal to appear.