Secondary Antibody Incubation for Western Blot

For the most widely used western blot method - indirect detection method, secondary antibody, which recognizes the primary antibody in Western Blot, is then incubated after primary antibody incubation. We have a variety of secondary antibodies available which can be used in different applications, including WB, ELISA, IP, IF/ICC and IHC.

Useful tips for secondary antibody incubation

The following are some useful tips about secondary 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

Why is a secondary antibody used in a Western Blot?

Through the indirect detection method, secondary antibodies are used to assist primary antibody to detect the protein of interest. Secondary antibodies are generally conjugated with fluorescent dye or an enzyme, whereas primary antibodies are labeled instead in the direct detection.

A wide variety of labeled secondary antibodies can be used for western blot detection. The choice of secondary antibody rely on the species of animal in which the primary antibody was raised (the host species). For example, if the primary antibody is a mouse monoclonal antibody, the secondary antibody must be an anti-mouse antibody acquired from a host other than the mouse.

How long should you incubate with secondary antibody in a Western Blot?

Usually 1-2 hours at room temperature or overnight at 4°C , with agitation.
After incubating with the secondary 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 secondary antibody.

Is it OK to perform secondary antibody incubation overnight in a Western Blot?

Incubation with secondary antibodies for one hour is a universally used standard protocol in western blotting. In contrast to common belief, interactions between primary antibodies and secondary antibodies are not always easy and fast. In some cases, a plateau detection limit is not reached even after 48 hours incubation, and a 16 hour incubation is needed for GAPDH to reach its plateau. Again, no increase in background is observed with longer incubations.

What is the recipe of secondary antibody dilution buffer in a Western Blot?

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%).

How do you dilute secondary antibody to the working concentration in a Western Blot?

Refer to the the manufacturer's datasheet and try a range of dilutions according to the suggested dilution. Then, optimize the dilution on the basis of the results.