Enzymatic labels are most commonly used for Western blotting and, although they require extra steps, can be extremely sensitive when optimized with an appropriate substrate. Horseradish peroxidase (HRP) is the enzymes used most extensively as labels for protein detection. An array of chromogenic, fluorogenic and chemiluminescent substrates are available for use with the enzyme. Horseradish peroxidase (HRP) conjugated antibodies are considered superior to antibody-AP conjugates with respect to the specific activities of both the enzyme and antibody due the smaller size of HRP enzyme and compatibility with conjugation reactions. In addition, the high activity rate, good stability, low cost and wide availability of substrates make HRP the enzyme of choice for most applications.
Enzyme conjugated antibodies offer the most flexibility in detection and documentation methods for Western blotting because of the variety of substrates available. The simplest detection/documentation system is to use chromogenic substrates. While not as sensitive as other substrates, the chromogenic substrates allow direct visualization of blot development. Unfortunately, chromogenic substrates tend to fade as the blot dries or when stored making the blot itself an unreliable means of documentation. However, it is fairly straightforward to either photocopy or directly scan the blot in order to make a permanent replica of chromogenic Western blot results.
HRP Western Blot Protocol - Critical Steps in HRP-Western Analysis
Prepare samples and determine protein concentrations.
Separate protein samples by SDS-PAGE.
Transfer to a membrane.
Blocking: Prepare blocking buffer in a tray. Remove the membrane from the Western blotting apparatus and soak in blocking buffer. Shake for 2 hour at room temperature. Discard the blocking buffer and wash twice with an adequate quantity of fresh wash buffer..
Primary antibody incubation: Dilute primary antibody to an appropriate concentration with blocking buffer.
HRP-labeled secondary antibody: Soak the membrane in secondary antibody diluted with blocking buffer and shake for 2 hour at room temperature. Rinse the membrane breifly three time with an adequate quantity of fresh wash buffer.
Detection the signal:Detection using a CCD camera.