1. For protein samples diluted with TBS and PBS, a good dilution method is determined by the antigen dilution concentration in the sample, but because the antigen concentration is unknown, it is necessary to perform a wide range of dilutions. The detection sensitivity of the SuperSignal West Pic chemiluminescent substrate can reach picogram level, so the dilution range of the sample can be from microgram level to picogram level. If too much antigen is used, the following will occur: non-specific bands, fuzzy bands, and reduced signal.
2. Prepare the transfer film. The number of membranes required depends on how many primary and / or secondary antibodies are blocked
Different dilution concentrations. Generally, the dilution of one or two primary antibodies is measured with two or three different dilutions of secondary antibodies. For example: 1/1000 primary antibody and 1/50000 secondary antibody; 1/1000 primary antibody and 1/100000 secondary antibody; 1/5000 primary antibody and 1/50000 secondary antibody; and 1/5000 and 1 / 100000 secondary antibody.
3. Place the membrane on the filter paper. Spot the antigen dilution on the membrane. Use the smallest possible amount of diluent on the membrane (2-5 ul is appropriate), because the larger the volume, the more diffuse the signal. The antigen solution is dried on the membrane for 10-30 minutes or until there is no visible moisture.
4. Block non-specific spots on the membrane with 0.05% Tween-20 blocking solution, and incubate for 1 h at room temperature with shaking.
5. Dilute the primary antibody into the blocking solution / Tween-20 detergent and add it to the membrane. Incubate at room temperature with shaking for 1 hour.
6. Wash the membrane 4-6 times with TBS or PBS, use the largest volume of eluent, and add 0.05% Tween to the eluent to reduce non-specific background. During each elution, suspend the membrane in the eluent and shake for approximately 5 minutes. Decant the eluent and repeat. Before incubation, a short rinse in the eluent may increase the efficiency of elution.
7. Prepare the secondary antibody / HRP conjugated blocking reagent / Tween-20 detergent dilution, add the secondary antibody dilution to the membrane and incubate for 1 h with shaking.
8. Follow Step 6 to clean the membrane again.
9. Prepare substrate working buffer, mix equal volume of luminol / enhancer and stable peroxide solution. Prepare enough volume to ensure that the blots are completely wet, and ensure that the blots will not dry out during the incubation process. Recommended volume: 0.1 ml / cm2 blotting surface.
10. Incubate the membrane for 5 minutes in the super-sensitivity substrate working solution;
11. Remove the film from the substrate and place it on plastic cloth or other protective film;
12. Attach the blot to the film and expose it next to the protein. Any standard or enhanced autoradiographic film can be used. It is recommended that the first exposure is 30-60 s. For best results, the exposure time can be different. In addition, CCD cameras or other imaging facilities may be used; however, these facilities may require longer exposure times.
13. On the best blotting membrane, the signal generated by the super-sensing reaction substrate may last for more than 8 h. Without the best results, the blot can be repeatedly exposed to the film or imaging system. As the imprinting time increases, the exposure time needs to be extended. If the best results are not obtained, this procedure can be repeated with antigen and / or antibody dilutions.
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