Immunodetection

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Overview

Immunodetection (immunological detection) is used to identify specific proteins blotted to membranes. This section provides an overview of immunodetection methods, workflow, protocol, and troubleshooting tips.

Related Topics: Total Protein Detection, Imaging and Analysis.

Immunodetection Workflow

The steps used for immunological detection vary little and are summarized in the western blotting workflow below.

After the proteins have been transferred to the membrane, the membrane is blocked, incubated with a primary antibody, washed, incubated with a secondary antibody, and washed again. The primary antibody is specific for the protein of interest and the secondary antibody enables its detection. The secondary antibody can be radiolabeled, labeled with a fluorescent compound or gold particles, or conjugated to an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP) for subsequent detection.

For many years, radiolabeled secondary antibodies were the method of choice for detection, but newer detection reagents have enabled less hazardous and more user-friendly methodologies, while maintaining the same degree of sensitivity. Available detection methods now also include colorimetric, chemiluminescence, fluorescence, bioluminescence, chemifluorescence, and immunogold detection.

Immunodetection Workflow

Western blotting workflow.

Blocking

Following transfer, unoccupied binding sites on the membranes must be blocked to prevent nonspecific binding of probes; failure to completely block these sites can lead to high background. A variety of blocking reagents are available, including gelatin, nonfat milk, and bovine serum albumin (BSA), which are compared in the table below. The detection system can be optimized for minimal background with no loss of signal by testing several reagents. The type of membrane also affects the selection of the blocker.

Comparison of blocking reagents.

Blocking Reagent Membrane Compatibility Recommended Concentration Notes
Gelatin Nitrocellulose 1–3% Requires heat to solubilize
Nonfat dry milk, BLOTTO, blotting-grade blocker Nitrocellulose, PVDF 0.5–5% PVDF requires higher concentrations of nonfat milk than nitrocellulose
Bovine serum albumin (BSA) Nitrocellulose, PVDF 1–5% PVDF requires higher concentrations of BSA than nitrocellulose
Tween 20 Nitrocellulose 0.05–0.3% May strip some proteins from the blot

Antibody Incubations

A typical immunodetection experimental system utilizes two rounds of antibody incubation:

  • The primary antibody, which is directed against the target antigen; the antigen may be a ligand on a protein, the protein itself, a specific epitope on a protein, or a carbohydrate group
  • The secondary antibody, which recognizes and binds to the primary antibody; it is usually conjugated to an enzyme such as AP or HRP, and an enzyme-substrate reaction is part of the detection process (see figure below)

Antibody Incubations

Specific enzymatic detection of membrane-bound antigens.

Antibody incubations are generally carried out in antibody buffer containing Tris-buffered saline with Tween (TTBS) and a blocking reagent.

Washes

Washing the blot between the two antibody incubations and prior to detection removes excess antibody and prevents nonspecific binding. Although washing solutions and times may vary (depending on the antibodies and detection systems used), washes generally involve Tris-buffered saline (TBS) or phosphate-buffered saline (PBS). A detergent such as Tween 20 may be added to decrease background, but it may also adversely inhibit certain detection reactions (see the instruction manuals for details).

Troubleshooting

Immunodetection

Problem Cause          Solution
Overall high background Blocking was incomplete
  • Increase the concentration of blocker
  • Increase the duration of the blocking step
  • Use a different blocking agent
Blocker was impure. Nonfat dry milk was not pure. The blocker may be contaminated with material that nonspecifically binds probes
  • Use a pure protein such as BSA or casein as a blocker
Wash protocols were insufficient
  • Increase the number, duration, or stringency of the washes
  • Include progressively stronger detergents in the washes; for example, SDS is stronger than Nonidet P-40 (NP-40), which is stronger than Tween 20
  • Include Tween 20 in the antibody dilution buffers to reduce nonspecific binding
The blot was left in the enzyme substrate too long (colorimetric detection)
  • Remove the blot from the substrate solution when the signal-to-noise level is acceptable, and immerse in diH2O
Contamination occurred during electrophoresis or transfer
  • Discard and prepare fresh gels and transfer solutions
  • Replace or thoroughly clean contaminated foam pads if a tank blotter was used
Excessive amounts of protein were loaded on the gel or too much SDS was used in the transfer buffer. Proteins can pass through the membrane without binding and recirculate through a tank blotting system
  • Reduce the amount of protein on the gel or SDS in the transfer buffer
  • Add a second sheet of membrane to bind excess protein
The primary or secondary antibody was too concentrated
  • Increase antibody dilutions
  • Perform a dot-blot experiment to optimize working antibody concentration
Incubation trays were contaminated
  • Clean the trays or use disposable trays
Nonspecific reactions between bound proteins and probes The primary or secondary antibody was contaminated with nonspecific IgG or with IgG cross-reactivity among species
  • Use purified IgG primary antibody fractions and affinity-purified blotting-grade cross-adsorbed secondary antibody
Monoclonal antibodies reacted nonspecifically with SDS-denatured proteins
  • Compare the binding of other monoclonal or polyclonal antibodies
  • Blot native proteins as a comparison
Nonspecific interactions are occurring due to ionic associations. For example, avidin, a glycosylated protein, may bind to more acidic proteins on blots
  • Increase the ionic strength of the incubation buffers
  • Increase the number, duration, or stringency of the washes
  • Include progressively stronger detergents in the washes; for example, SDS is stronger than Nonidet P-40 (NP-40), which is stronger than Tween 20
  • Include Tween 20 in the antibody dilution buffers to reduce nonspecific binding
No reaction or weak signal The sample load was insufficient
  • Increase the amount of protein applied
  • Concentrate the sample prior to loading
The detection system was not working or is not sensitive enough
  • Use a more sensitive assay system
  • Include proper positive and negative control antigen lanes to test for system sensitivity; consult manual
Proteins were washed from the membrane during assays
  • Reduce the number of washes or reduce the stringency of washing conditions during subsequent assay steps
Antigen binding to the membrane was insufficient
  • Stain the blot after transfer or use prestained standards to assess transfer efficiency. Alternatively, use stain-free technology to assess sample binding on the blot. See the previous section for suggestions on improving transfer-related problems
Antigen denaturation occurred during electrophoresis or transfer
  • Antibodies, especially monoclonals, may not recognize denatured antigens
  • Electrophorese and transfer proteins under native conditions. Use a cooling coil and a refrigerated recirculating bath to transfer heat-sensitive proteins
Epitope was blocked by total protein stain
  • Some total protein stains (such as amido black and colloidal gold) interfere with antibody recognition of the antigen. Do not use a total protein stain or use a different stain or stain-free technology
The primary or secondary antibody was inactive or nonsaturating
  • Store the reagents at recommended conditions. Avoid repeated freeze-thaw cycles, bacterial contamination, and heat inactivation
  • Detergents may affect the binding of some antibodies. Eliminate them from the assay, except for the wash after blocking
  • If the antibody titer is too low, optimize the concentration using a dot-blot experiment
  • Increase the antibody incubation times
The enzyme conjugate was inactive or nonsaturating
  • Test the reagent for activity*
  • Store the reagents at recommended conditions. Avoid repeated freeze-thaw cycles, bacterial contamination, and heat inactivation
  • Sodium azide is a potent inhibitor of horseradish peroxidase. Use a different biocide such as gentamicin sulfate
  • Undistilled water may cause inactivation of the enzyme. Use only ddH2O
  • If the conjugate concentration is too low, optimize using a dot-blot experiment
The color development reagent was inactive
  • Test the reagent for activity* and remake if necessary
Regions of poor or uneven signal during detection The membrane was allowed to dry during handling
  • High intensity or rapid transfer methods generate heat. Ensure that warm membranes are not allowed to dry after transfer

* Tests for Monitoring Reagent Activity

  1. Test the activity of the color development solution. Combine 1.0 ml of the color development solution with 10 µl of full-strength secondary antibody conjugate. The color reaction should occur immediately. If color fails to develop within a few minutes, the color development solution is inactive. Prepare a fresh working solution and repeat the color development assay.
  2. Test the activity of the conjugate solution. Combine 1.0 ml of the color development solution tested above and 1.0 ml of the 1:3,000 dilution conjugate solution. A light-blue tinge should develop within 15 min. If color fails to develop within 25 min, the conjugate solution is suspect. Repeat the procedure with a freshly prepared dilution of conjugate.
  3. Test the activity of the first antibody solution. Use an ELISA, RID, Ouchterlony immunodiffusion, or precipitation test to determine reactivity of the antibody with the antigen. If possible, repeat the assay procedure with a more concentrated primary antibody solution.

 

Related Content

Literature
Number Description Download
2895 Protein Blotting Guide, Interactive PDF, Rev B Click to download
3179 Fluorescent Nanoparticles for Western Blotting, Rev A Click to download
5723 Increase Western Blot Throughput with Multiplex Fluorescent Detection, Rev B Click to download
5809 Imaging of Chemiluminescent Western Blots: Comparison of Digital Imaging and X-ray Film, Rev A Click to download
5881 Enhanced Multiplex Fluorescent Western Blotting, Rev A Click to download
Number Description Options
6216 Detection Buffer Formulations Click to download
6218 Blot Stripping & Reprobing Click to download
6219 Immunodetection Click to download
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