High sensitivity from signal amplification and detection of multiple epitopes. Produced from differing B-cell lineages – recognize multiple epitopes on the antigen. Possible lower sensitivity due to reduced binding affinity for epitopes compromised by sample preparation/electrophoresis. High specificity, batch-to-batch consistency and are often well characterized. Produced from a single B-cell lineage – specific to one epitope on an antigen. Advantages and disadvantages of Monoclonal and Polyclonal antibodies. Both types have benefits and limitations (which are shown in the table below). Whereas polyclonal antibodies are produced from differing B-cell lineages and so contain multiple clones recognizing different epitopes on the antigen. Monoclonal antibodies are produced from a single clone of B cells and produce antibodies specific to one epitope. Monoclonal and Polyclonal Primary Antibodiesīoth monoclonal and polyclonal antibodies can be used for western blotting. This can be particularly problematic when the protein of interest has a molecular weight that is similar to either that of the heavy chain (50 kDa) or the light chain (25 kDa), as differentiation between the protein of interest and endogenous mouse IgG becomes impossible. If the primary antibody is also made in mouse, an anti-mouse secondary antibody will detect not only the primary antibody binding to the protein of interest but also to the heavy and light chains of the immunoglobulin in the mouse sample. For example, a sample from mouse may contain mouse immunoglobulins. This is to avoid the secondary antibody detecting immunoglobulins from the sample if present. Ideally, the primary antibody should be raised in a different species than the sample to be analyzed. This is especially true of monoclonal antibodies. Some antibodies raised using native protein will only recognize the epitopes on the surface of a protein in its native, oxidized conformation. If purified proteins are used as an immunogen, the antibodies generated against them may not recognize the linearized epitopes of the denatured proteins in western blotting. In those cases, peptides generated to the regions of difference increase the possibility of making a differentiating antibody. Peptides can also be useful as immunogens when an antibody needs to differentiate between two proteins having highly conserved amino acid sequences. Therefore, antibodies generated using peptides can be useful reagents for western blotting as target proteins have linearized epitopes due to denaturation and reduction during sample processing. In addition, unless added to the peptide during synthesis, they do not exhibit any post-translational modifications. Synthetic peptide immunogens rarely fold into the comparable tertiary structure of the native protein. Care should be taken to select antibodies raised against well-characterized immunogens. Choosing an antibody raised against an unsuitable or poor quality immunogen will lead to unreliable and potentially irreproducible results. The type of immunogens used to produce the antibody dramatically influences the specificity of the antibody for the target protein. There is a wide range of primary antibodies available commercially in different formats, clonality and specificity, which are important factors to consider when selecting an appropriate reagent. This maximizes the sensitivity of this technique and increases the signal-to-noise ratio.Ĭonsiderations for Selecting a Primary Antibody Following incubation, with the primary antibody the membrane is washed to remove any excess and unbound antibody. The membrane is incubated with the primary antibody at room temperature (1 hr is commonly used) on a rocker or shaking platform, although overnight incubations at 4☌ may also be effective. Added after membrane blocking, the primary antibody binds specifically to the protein of interest. The primary antibody is the reagent used to detect the protein of interest. Reprobing and Stripping for Additional Target Proteins.Monoclonal and Polyclonal Primary Antibodies.Considerations for Selecting a Primary Antibody.
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