Quantification of Protein in Brain Tissue by Western Immunoblot Analysis - Drugs of Abuse: Neurological Reviews and Protocols - page 264

Fig. 3. Western blot of primary striatal neurons, developed with a phosphorylation-
specifi c cAMP response element binding protein (CREB) antibody. Treatment of
the cultures for 15 min with either glutamate, N -methyl- D -aspartate, or dopamine
leads to the phosphorylation of the transcription factor CREB at 133 Ser. The blot was
developed with an antibody that detects CREB only when it is phosphorylated at the
133 Ser phosphorylation site.
9. Too little or too much staining: Weak staining can be caused by (a) insuffi cient
concentration of primary or secondary antibody; (b) the absence of antigen in
the sample, or the concentration of the antigen is below the detection level;
or (c) problems with the transfer. There are several solutions to resolve the
problem. First, various concentrations of antibody both higher and lower dilutions
can be tried. Second, the experimental protocol can be validated with either
commercially available antigen or in an experimental system that has been
reported to give good results; for example, retaining phosphorylation in in vivo
experiments can be diffi cult, and phosphorylation-specifi c antibodies may have
not antigens to react with. When using phosphorylation-specifi c antibodies, in
vitro phosphorylation of the antigen as a positive control can be considered.
Third, if the antigen in the sample is too low, immunoprecipitation before
running the Western blot can be performed. Make sure the antibody used for
immunoprecipitation is from a different host than the antibody used for the
Western blot. Finally, make sure (a) there are no air bubbles between the gel and
the membrane in the transfer, (b) the electrotransfer has been set up correctly
and in the correct orientation (Fig. 1) , (c) to use enough current and time for the
transfer; you can use prestained protein size markers, which are available from
commercial sources, to monitor the transfer
10. High background may be caused by: (a) antibodies used are too concentrated,
(b) blots are not washed enough, (c) blocking solutions and blocking times
are insufficient, (d) protein samples are overloaded, and (e) exposure time
with substrate is too long. If high background happens, one can (a) try lower
concentrations of antibody (higher dilutions), lower temperatures during incuba-
tion (e.g., 4°C) and lower exposure times to primary antibody; (b) increase
washing time and number of washes; try washing after the primary antibody
overnight at 4°C with slightly shaking; (c) try different blocking solutions and
different detergents in the blocking solution; add blocking solution to primary