After being blocked, the western blot membrane is subsequently incubated with a primary antibody - recognition of the target protein. The choice of a primary antibody for a Western blot will depend on the antigen to be detected and what antibodies are available to that antigen. Alternatively, a primary antibody should be made to recognize the antigen of interest. Both monoclonal and polyclonal antibodies work well for western blotting. Because of their specificity, purity and consistency, monoclonal antibodies are valued, resulting in lower background. Polyclonal antibodies are less expensive and less time-consuming and often with a high affinity for the antigen. Serum or ascites fluid and other crude antibody preparations are some used for western blotting. However, the impurities present may increase background. To get antibodies with the greatest specificity, they can be affinity-purified using the immobilized antigen.
Western blot primary antibodies contain both monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). In Western Blot, according to different purposes, different types of primary antibodies can be chosen. We have a wide range of primary antibodies available for your choice.
The following are some useful tips about primary antibody incubation, hoping it can help you solve some problems in Western Blotting. In addition, if you have any issues in your Western Blotting, please feel free to contact firstname.lastname@example.org.
Antibodies are critical to the success of the western blot technique. In the indirect detection method, the primary antibody is used to specifically bind the protein of interest and then a labeled secondary antibody is used for detection; Through the direct detection method, the primary antibody is labeled with fluorescent dye or an enzyme, and it's responsible for both binding and detection of the protein of interest.
It depends on the binding affinity of the antibody to the target protein, usually 2 hours at room temperature incubation or 4°C incubation overnight.
After incubating with the primary antibody, the membrane is then washed with TBST or PBST buffer for 4-5 times, about 5 minutes each time with agitation, to remove residual unbound primary antibody.
It depends. Normally, you can increase the primary antibody incubation time but it will cause non-specific bands and high background. However, sometimes, it is recommended that a more dilute antibody, and a prolonged incubation with agitation might ensure specific binding and adequate homogenous covering of the membrane.
1X TBS, 0.1% Tween-20 with 5% BSA; For 20 ml, add 2 ml 10X TBS to 18 ml water, mix. Add 1.0 g BSA and mix well. While stirring, add 20 μl Tween-20 (100%).
Dilute the primary antibody with fresh blocking buffer to the recommended dilution/concentration according to the manufacturer's datasheet and determine the optimal working concentration. For example, if a product datasheet suggests using a 1:1000 dilution for Western blotting, it is recommended to make serial dilutions of 1:250, 1:500, 1:1000, 1:2000 and 1:4000. This should determine the optimal dilution for your individual sample conditions.
The blocking buffer containing Tween-20 can block hydrophobic interactions. In the absence of that, you may get random, non-specific binding. While some other laboratories use TBST instead of blocking buffer. The results are variable from antibody to antibody.