It is sometimes necessary to detect more than one protein by chemiluminescence on the same Western blot membrane. One way to detect multiple proteins is by stripping and reprobing the membrane with a different primary antibody.

Stripping the membrane involves harsh conditions to disrupt the interaction between the membrane-bound protein and the primary antibody. This process enables reprobing with new primary antibody for further protein identification.

Although the method of stripping and reprobing risks loss of protein from the membrane, not having to perform a second Western blot does save time and minimizes use of valuable protein samples.

Careful consideration of the stripping conditions can help minimize the risk of protein loss from the membrane. These considerations include using combinations of detergents, reducing agents, heat, and high or low pH.

Stripping membranes using heat and detergent

A combination of heat and detergent is one option for stripping Western blot membranes.

In this method, the prewashed membrane is incubated with stripping buffer that contains 2% sodium dodecyl sulfate (SDS), 100 mM β-mercaptoethanol in 62.5 mM Tris-HCl, pH 6.8 for 30 min at 50°C to 70°C.

The optimal stripping temperature requires testing on an antibody-by-antibody basis. Dot-blotting is one method of quickly testing the conditions, but it requires additional protein that might not be available. If there are limited quantities of protein, then in some cases the original membrane can be cut into strips instead.

Low pH

An alternative method of membrane stripping is to use a low pH.

The membrane is soaked in a stripping buffer of 25 mM glycine-HCl, pH 2, supplemented with 1% SDS, for approximately 30 min under constant agitation.

If the membrane is not completely stripped, the incubation time can be increased up to 1 h. However, using an acidic stripping buffer for a prolonged period does carry an increased risk of protein loss.

When using chemiluminescence, a milder protocol involves incubation in 0.2 M glycine (pH 2.8) at room temperature for 30 min, followed by two washes in TBS-Tween™ or PBS-Tween for 10 min each.

High pH

High pH solutions are also used for membrane stripping.

The high pH method involves two incubations in 0.2 M NaOH for 5 min, followed by a minute wash with water. If signal from the original antibody remains, the concentration of NaOH can be raised to 2 M and the incubation time to 30 min.

It might be necessary to reblock the membrane following this process, depending on the NaOH concentration and incubation time.

High salt solutions

Using a high salt concentration is another effective solution for stripping Western blot membranes.

The membrane is soaked in PBS or TBS buffer supplemented with 0.5 M NaCl and 0.2% SDS for between 30 min and 2 h. The blot is rinsed with water and, depending on the soaking time, might need reblocking before reprobing.

Alternative methods of detecting additional proteins

Sequential labeling with ECL detection

When using enhanced chemiluminescence (ECL) detection for a Western blot, a sequential labeling method is available for quick detection of a second protein on a single membrane.

Labeling and detection of the first protein is performed as normal using ECL. The horseradish peroxidase (HRP) is then inactivated (quenched) using hydrogen peroxide (H2O2) and the membrane is washed. As a result, the second protein can be labeled with a different antibody for detection without any interference.

In the majority of cases, a 30 min incubation with 15% H2O2 is sufficient to fully quench the signal from HRP-labeled protein. However, the amount of HRP present does influence the concentration of H2O2 necessary, as well as the incubation time.

Multiplex detection

To avoid stripping and reprobing altogether, multifluorescence (multiplex) detection can be used to detect multiple proteins on the same membrane. In this technique, secondary antibodies labeled with fluorophores enable simultaneous detection of more than one protein.

Amersham ECL Plex provides the tools for this method, which has the added benefit of no protein loss risk.

For more hints and tips, read our stripping and reprobing blog post. It includes an approach for determining the optimal stripping strategy with examples.

Download the Western blotting principles and methods handbook how-to hints and tips, protocols, and much more.