For a given antigen, the antibody concentration of Jia depends on the antigen and the antibody itself. The affinity of the antigen and the antibody, and the specific response of the primary and secondary antibodies are different. You antigen and antibody concentrations can be determined by immunoblot experiments using different concentrations of antigen and antibody. In addition, a faster and simpler method is to perform dot blot experiments. Here's how to do a dot blot using a super sensitive signal substrate.
Note: All antibodies are diluted to an initial starting concentration of 1 mg / mL.
1. For protein samples diluted with TBS and PBS, a good dilution method is determined by the concentration of the antigen in the sample. However, since the concentration of the antigen is unknown, it is necessary to perform a wide range of dilution tests. The detection sensitivity of SuperSignal West Pic chemiluminescence substrate can reach pico-g level, so the sample can be diluted from microgram level to pico-gram level. If too much antigen is to be used, the result is the following: non-specific bands, blurred bands, and reduced signal.
2. Prepare the transfer film. The number of membranes required depends on how many different dilution concentrations of the primary and / or secondary antibody are shielded. Generally, the dilution of one or two primary antibodies is determined using two or three different secondary antibody dilutions. 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 dilution on the membrane (2-5 ul is appropriate), because the larger the volume used, the more diffuse the signal. The antigen solution is allowed to dry on the membrane for 10-30 minutes or until there is no visible moisture.
4. Block the 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 blocking solution / Tween-20 detergent and add it to the membrane. Incubate with shaking at room temperature for 1 h.
6. Wash the membrane 4-6 times with TBS or PBS. Use the largest possible volume of eluent. Add 0.05% Tween to the eluent to reduce non-specific background. During each elution, the membrane was suspended in the eluent and shaken for about 5 minutes. The eluate was decanted and repeated. A short rinse in the eluent prior to incubation may increase elution efficiency.
7. Prepare a secondary antibody / HRP-bound blocking reagent / Tween-20 detergent dilution. Add the secondary antibody dilution to the membrane and incubate for 1 h.
8. Clean the membrane again as described in step 6.
9. Prepare substrate working buffer, mix equal volumes of Luminox / Enhancer and stable peroxide solution. Prepare enough volume to ensure that the blots are completely moistened and that the blots do not dry out during incubation. Recommended volume: 0.1 ml / cm2 blotted surface.
10. Incubate the membrane in the working solution of the super-sensitive chromogenic substrate for 5 minutes;
11. Remove the film from the substrate and place it on a plastic cloth or other protective film;
12. Place the blot on the film and expose next to the protein. Any standard or enhanced autoradiographic film can be used. The recommended exposure is 30-60 s. To get Jia results, the exposure time can be different. Alternatively, CCD cameras or other imaging facilities may be used; however, these facilities may require longer exposure times.
13. On Jia's imprinted membrane, the signal generated by the super-induction reaction substrate may last more than 8 h. No Jia results were obtained, and the blot could be repeatedly exposed to film or imaging systems. As the blotting time increases, you need to increase the exposure time. If no Jia results are obtained, the procedure can be repeated with antigen and / or antibody dilutions.
Keywords: ELISA kit
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