Protein analysis

Protein detection: Specific antibody-mediated detection of proteins on a membrane

Antibodies are proteins synthesized by an animal in response to the presence of a foreign substance (antigen). By injecting an antigen into an animal, after a certain time, antibodies of a class termed IgG (immunoglobulin G) that react specifically with the introduced protein can be harvested from the animal’s serum. Each antibody has a specific affinity for a particular region of the antigen.

This region is termed an epitope. The antibody–epitope interaction can be utilized for highly specific and sensitive detection of a protein that has been immobilized on a membrane, in a process termed immunodetection. The antibody that binds specifically to the protein of interest is termed the primary antibody and is often obtained from rabbits or mice. The primary antibody is applied to the membrane and allowed to bind to the target protein. In order to locate the primary antibody (and therefore the protein of interest), a secondary antibody is required. The secondary antibody recognizes and binds to all IgG antibodies from another animal species. It is important that the secondary antibody used in an experiment is directed against IgGs from the species of origin of the primary antibody.

For example, if the primary antibody was generated in a mouse, then a goat anti-mouse secondary antibody can be used for detection. The secondary antibody is usually chemically coupled to a reporter, which allows detection and visualization of the antibody. Fluorescing molecules, or enzymes that produce colored or luminescent reaction products, are typically used as reporter groups. A primary antibody chemically coupled to a reporter enzyme is termed a conjugate, and can be used for direct detection without the use of a secondary antibody.
After protein transfer from an SDS-PAGE gel to a membrane (see Western transfer), the remaining protein-free sites on the membrane must be blocked. This prevents the primary or secondary antibody from binding directly to the membrane and giving rise to a high background signal. Several blocking reagents are in common use, including nonfat dried milk, BSA, and casein. After blocking, the primary antibody is added and allowed to bind to the protein (see figure Immunodetection of a protein immobilized on a membrane). After washing (which removes nonspecifically bound antibody), the secondary antibody is added, to detect where the primary antibody has bound. After another wash step, the location of the secondary antibody, (and therefore the primary antibody and the protein of interest) is determined by adding a substrate for the enzyme conjugated to the secondary antibody. Substrates are available that give rise to a colored compound (chromogenic detection), or to the emission of light (chemiluminescent detection), at the reaction site.

The use of an antibody that reacts specifically with an epitope commonly introduced into a recombinant protein eliminates the need for a protein-specific antibody, and allows the use of one antibody for the detection of all proteins containing this feature. Coupling a reporter enzyme directly to such antibodies eliminates the need for a secondary antibody, and delivers significant time savings. Detailed information on immunodetection procedures can be found in current molecular biology manuals (3, 4).
Immunodetection of a protein immobilized on a membrane

Materials required

  • Western blot (see Western transfer)
  • TBS buffer (see table TBS buffer)
  • TBS-Tween/Triton buffer (see table TBS-Tween/Triton buffer)
  • Blocking buffer (see table Blocking buffer)
  • Primary (protein-specific) antibody stock solution
  • Secondary antibody-enzyme conjugate stock solution
  • Secondary antibody dilution buffer (see table Secondary antibody dilution buffer)
  • Chemiluminescent substrate

Note: For chemiluminescent detection, BSA does not sufficiently block nonspecific binding of the secondary antibody to the membrane, so milk powder should be used to dilute the secondary antibody. However, in some cases, dilution of antibody in a buffer containing milk powder can lead to reduced sensitivity. If this is the case, the primary antibody should be diluted in BSA solution, and the secondary antibody in milk powder solution.

TBS buffer
Composition of working solution, pH 7.5 Component   Amount per liter
10 mM Tris·Cl Tris base 1.2 g
150 mM NaCl NaCl 8.8 g

TBS-Tween/Triton buffer
Composition of working solution, pH 7.5 Component Amount per liter
20 mM Tris·Cl Tris base 2.4 g
500 mM NaCl NaCl 29.2 g
0.05% (v/v) Tween 20 Tween 20 500 µl
0.2% (v/v) Triton X-100 Triton X-100 2 ml

Blocking buffer
Composition of working solution Component Amount per liter
3% (w/v) BSA in TBS buffer BSA* dissolved in TBS buffer 30 g
Alternative: 1% (w/v) alkali-soluble
casein in TBS buffer
Alkali-soluble casein dissolved
in TBS buffer
10 g

Secondary antibody dilution buffer
Composition of working solution Component Amount per liter
3% (w/v) BSA in TBS buffer BSA* dissolved in TBS buffer 30 g
Alternative: 1% (w/v) alkali-soluble
casein in TBS buffer
Alkali-soluble casein dissolved
in TBS buffer
10 g

Perform all incubation and wash steps on a rocking platform or orbital shaker.

  1. Wash membrane twice for 10 min each time with TBS buffer at room temperature.
  2. Incubate membrane for 1 h in blocking buffer at room temperature.

    Tip: Seal the vessel used for incubation with plastic film to prevent the membrane from drying out.

  3. Wash membrane twice for 10 min each time in TBS-Tween/Triton buffer at room temperature.
  4. Wash membrane for 10 min with TBS buffer at room temperature.
  5. Incubate membrane with primary antibody solution (1/1000–1/2000 dilution of primary antibody stock solution in blocking buffer) at room temperature for 1 h.

    Tip: Make sure that the membrane is fully coated by the antibody solution. Do not allow the membrane to dry out.

    Tip: To reduce the volume of antibody required, the membrane can be sealed in a plastic bag.

  6. Wash membrane twice for 10 min each time in TBS-Tween/Triton buffer at room temperature.
  7. Wash membrane for 10 min in TBS buffer at room temperature.
  8. Incubate the membrane with a dilution of secondary antibody in 10% nonfat dried milk in TBS for 1 h at room temperature. Dilute the secondary antibody according to the manufacturer’s recommendations.

    Tip: Ensure that your secondary antibody is directed against the species of origin of your primary antibody!

    Tip: Milk powder is needed to reduce background because BSA does not block sufficiently for the very sensitive chemiluminescent detection method.

    Tip: Use the lowest recommended concentration to avoid false signals.

  9. Wash 4 times for 10 min each time in TBS-Tween/Triton buffer at room temperature.
  10. Perform chemiluminescent detection reaction, cover the membrane with thin plastic wrap, and expose to X-ray film according to the manufacturer’s recommendations.

    Tip: Ensure that you use the correct chemiluminescent detection substrate, i.e., an AP substrate for AP conjugates, or an HRP substrate for HRP conjugates!

    Tip: Blots can be wrapped in plastic wrap and stored at 4°C. Protocols exist for stripping the blot, which can subsequently be reprobed with a different antibody (5).

Materials required

  • Western blot (see Western transfer)
  • TBS buffer (see table TBS buffer)
  • TBS-Tween/Triton buffer (see table TBS-Tween/Triton buffer)
  • Blocking buffer (see table Blocking buffer)
  • Primary (protein-specific) antibody stock solution
  • Secondary antibody-enzyme conjugate stock solution
  • Chromogenic substrate (see table Chromogenic substrates for immunoblotting procedures)

Note: 3% BSA (w/v) can be used as both the blocking buffer and the secondary antibody dilution buffer for chromogenic detection.

Chromogenic substrates for immunoblotting procedures
Composition of working solution   Component Amount per liter
10% (w/v) nonfat dried milk powder
in TBS buffer
Nonfat dried milk powder
dissolved in TBS buffer
100 g
Alternative: 1% (w/v) alkali-soluble
casein in TBS buffer
Alkali-soluble casein dissolved
in TBS buffer
10 g

Perform all incubation and wash steps on a rocking platform or orbital shaker.

  1. Follow steps 1–7 of the protocol Immunodetection using a chemiluminescent method
  2. Incubate the membrane with secondary antibody solution diluted in 3% BSA (w/v) in TBS for 1 h at room temperature. Dilute according to the manufacturer’s recommendations.

    Tip: Ensure that your secondary antibody is directed against the species of origin of your primary antibody.

    Tip: Use the lowest recommended concentration to avoid false signals.

  3. Wash 4 times for 10 min each time in TBS-Tween/Triton buffer at room temperature.
  4. Stain with AP or HRP staining solution until the signal is clearly visible (approximately 5–15 min). Do not shake blots during color development.

    Tip: Ensure that you use the correct chromogenic detection substrate, i.e., an AP substrate for AP conjugates, or an HRP substrate for HRP conjugates.

  5. Stop the chromogenic reaction by rinsing the membrane twice with water.
  6. Dry the membrane and photograph as soon as possible as the colors will fade with time. The product formed when using HRP is particularly unstable.
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