Blotting Procedural Guide

Choosing a Membrane

The choice of blotting matrix depends on physical properties such as sensitivity, binding capacity, stability of bound molecules and compatibility with the assay system.  Nitrocellulose is one of the most commonly used membranes due to its high affinity for proteins and cellular macromolecules.  In general, it offers high binding capacity and minimal background problems and is used in a wide variety of protein transfer techniques.  However, the brittle structure of nitrocellulose necessitates that it be handled carefully and stored in a cool area (0-5^oC).  Both nicrocellulose and PVDF are suitable for Western blotting.

Preparing the Membrane

To prepare nitrocellulose for blotting, it should be completely prewetted in distilled water or buffer.   PVDF is hydrophobic and requires prewetting with methanol prior to use.  Excess moisture is removed by laying the membrane on filter paper.  This procedure provides the membrane with uniform binding properties.  As the membrane can be blocked by skin oils and proteins, gloves should be wrorn and forceps used to handle the membrane.

Blocking the Membrane

Following the sample application, the membrane is blocked with an inert protein to reduce nonspecific binding and to protect adsorbed protein from surface denaturation.  This is done by completely immersing the membrane in protein solution for at least 10 to 15 minutes. A gentle rocking motion may enhance the binding rate in this and subsequent steps.  Blocking also serves as the washing function at this step.  KPL's Detector Block (Catalog No. 71-83-00) is a proprietary blocking agent that enhances sensitivity while minimizing nonspecific noise. BSA Diluent/Blocking Solution (Catalog No. 50-61-00) and Milk Diluent/Blocking Solution (Catalog No. 50-82-01) are also provided for this purpose and for diluting samples, controls and antibodies.

Controlling the Assay

Every procedure should include several controls to verify assay performance; positive controls assure that all reactants are performing correctly and negative controls help define background. Positive controls may be positive control antigen.  Negative controls are known negative reference samples that may be an irrrelevant primary antibody or control serum.  A background control consisting of all reagents except the sample should also be incorporated to help identify the cause of background if it is a problem.  

Optimizing Assay Conditions

Optimization experiments are critical to obtain high sensitivity and low background  This step is especially important in Western blotting protocols where antigen may be partially denatured during gel electrophoresis.  In dot blot assays the optimal concentrations of antigen and primary antibody are found by titrating the antigen in a series of two-fold dilutions of primay antibody solution.  In Western blots serial dilutions of the primary antibody are incubated with the transfer blots.The blots or strips are then immersed in a fixed concentration of labeled antibody followed by substrate solution.  After signal development, the strips are examined for intensity to determine the concentrations that give optimal signal-to-noise ratio.  In like manner the optimal dilution of the labeled antibody can be determined by performing serial dilutions of this reagent.  Incubation times and temperatures are variables that should also be optimized.  Room temperature is suitable for most procedures.  Uniform incubation times and temperature from assay to-assay will help assure consistent results.  Optimization becomes critical as the sensitivity of the substrate increases.  For instance, slight changes in antibody concentration may impact signal-to-noise dramatically when using high reactivity substrates like LumiGLO Reserve (Catalog No. 54-71-00).

Washing the Membrane

The membrane is washed after addition of the primary antibody and the conjugate.  The washing procedure reduces background color by removing unbound reactant from the membrane. KPL's Wash Solution (Catalog No. 50-63-00) contains 0.02% Tween-20, a wetting agent that minimizes nonspecific attachment of proteins to the membrane.  If the procedure is interrupted at any point, the membrane can remain in the wash solution until the assay is resumed.  Note: Prolonged soaking in wash solutions can significantly reduce phosphatase enzyme activity.

Amplifying the Reaction

The enzyme conjugated to the secondary antibody or streptavidin serves as a signal generator which demonstrates the presence of target protein when reacted with substrate.  Labeled antibodies bind to multiple sites on each antigen or primary antibody, resulting in signal amplification and high sensitivity.  Polyclonal antibodies are preferred for blotting as they contain mixtures of antibodies that bind to many sites on the antigen.  They are more likely than monoclonal antibodies to recognize partially denatured antigen or epitopes exposed upon denaturation.

Choosing the Optimal Blotting Substrate

Chromogenic and Chemiluminescent Substrates