Technical Tips: Immunochemical Applications |
1. What is the difference between the titer and the working dilution of an antibody? Top
The titer of an antibody refers to the lowest concentration (highest dilution) at which the antibody is still effective in a given system. The working dilution is usually the concentration which gives a good sensitivity with a high signal:noise ratio. Using too high a concentration is wasteful and may give rise to non-specific signals. On the other hand, using too low a concentration will lead to a loss in sensitivity.
2. Why do I observe diffuse or moderate to intense staining of several tissue elements? Top
Diffuse staining occurs when the specimen is fixed for too long resulting in masking of antigenic determinants due to aldehyde cross-linking and increased hydrophobicity of tissue. Use of trypsin or other proteolytic enzymes will breakdown the cross-linking and render some tissue antigens reactive. In other instances, the sections may be too thick or the specimens may contain crushed or necrotic elements. This leads to a false negative staining accompanied by intense background staining. On the other hand, using an insufficiently diluted primary antibody leads to an intense staining of specimen or positive control.
3. Serum from which species should be used as a blocking agent in immunochemical applications? Top
The serum used for blocking should not be from the same species as the source of the primary antibody. The best choice may be to use serum from the species of the source of secondary antibody.
4. What can I use to block non-specific binding in immunological techniques? Top
This will largely depend on the type of antibody used. When working with anti-phosphotyrosine one should not use milk as a blocking agent as milk contains higher concentration of phosphoproteins. Bovine serum albumin is one of the most common choices. Gelatin or serum (other than the species of primary antibody) may also be used to block non-specific binding.
5. What is meant by monospecific reactivity? Top
Monospecific denotes a term "having an effect only on a particular kind of cell or tissue containing a particular antigen". Hence, when a statement is made that an antibody is monospecific, it is to emphasize that it does not cross-react with any other antigen.
6. What is the advantage of using affinity absorbed antibodies? Top
Affinity absorption is a method of separation by affinity chromatography. This method may be used to remove unwanted antibodies from a preparation. The preparation of antibodies is passed through a column matrix containing antigens against which the unwanted antibodies are directed. The unwanted antibodies remain bound to the column and the effluent contains the desired, affinity purified, antibodies. Alternatively, a column matrix with desired antigen coupled to it can be used. In this case, antibody directed against the coupled antigen remains bound to the column and may be then eluted using a solution that disrupts antigen-antibody binding. Affinity purified antibodies exhibit lower backgrounds than unabsorbed antibodies.
7. Why does one see a very weak or no signal in Western blot detection of proteins? Top
There are several possible causes of this. In some cases the primary or the secondary antibody might have lost its activity during storage or by repeated freezing and thawing. ELISA, immunoprecipitation, or another appropriate immunochemical assay can be used to confirm the low antibody reactivity. In other cases the antibody may have low affinity. Here one can increase the incubation period and the concentration of primary antibody. Weak signal can also result from an inefficient transfer of antigen out of the gel and inefficient binding of antigen to the membrane. This is caused by insufficient contact or due to the presence of air bubbles between the gel and the membrane or due to an excess of SDS in the gel or buffer.
8. What's the difference between a linear epitope and a conformational epitope? Top
A linear epitope consists of about 6 to 10 adjacent amino acids on a protein molecule that is recognized by an antibody. In contrast, conformational epitope consists of amino acids that are not arranged sequentially. Here the antibody recognizes only the 3-dimensional structure. When a protein molecule folds into a three dimensional structure the amino acids forming the epitope are juxtaposed enabling the antibody to recognize the sequence.
Knowledge of the differences between linear or conformational epitope is of significance in immunological applications. In a denatured protein only the linear epitope may be recognized. Hence in protocols where a denatured protein is used, such as in Western blotting, an antibody that recognizes a linear epitope is preferred. Sometimes an epitope is inside the protein. The epitope is then inaccessible to the antibody in a nondenaturing protocol, such as immunoprecipitation. A conformational epitope, by definition, must be on the outside of the folded protein. An antibody that recognizes the conformational epitope is suitable for mild, non-denaturing procedures, such as IP or flow cytometry.
Optimally, an antibody that recognizes a linear epitope on the surface of a normally folded protein will work well in both nondenaturing and denaturing protocols.
9. Why does one see extra bands on Western blots? Top
When extra bands appear below that of the desired protein, the most likely explanation is that they originated from proteolytic breakdown of the desired protein. This can be prevented by treating the sample with protease inhibitors during tissue or cell sample preparation. Also, if the sample buffer does not contain sufficient SDS and/or reducing agent (DTT, 2-mercaptoethanol, BMS), the protein may not be fully dissociated into its subunits, reduced or denatured. Hence, extra bands may appear above the desired protein on the gel. Boiling the sample in sample buffer immediately prior to loading can reduce these noncovalent interactions or disulfide linkages. Use of an irreversible reducing agent such as TCEP, instead of DTT or â-mercaptoethanol, may also be helpful.
Sometimes, the bands are specific; for example, an antibody made against a peptide coupled to BSA may recognize traces of BSA in the sample. Homologous proteins may also be recognized by the antibody. When using a polyclonal serum, some bands may be due to the presence of antibodies produced as a result of animal's exposure to similar antigens in its life. It is best to run a normal serum control to determine the specificity of any antibody.
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