Peptide-Based Drug Design

62 Winkler and Campbell
3.3. Probing
The number of probing possibilities is huge and even includes testing the
activity of peptides on living cells (14) or bacteria (35). Herewedescribethe
use of labeled proteins such as antibodies. The labels described are horseradish
peroxidase, alkaline phosphatase, and biotin. However, in general, most
labeled proteins can be used according the method described in Subheading
3.3.1. Differences are in label-specific detection of the bound labeled
protein.
3.3.1. HRP-Labeled Proteins and Chemiluminescence
1. If the membrane is dry, wash twice for 10 min each with methanol (or ethanol).
Then wash three times with T-TBS or T-PBS for at least 5 min each.
2. Blocking: To decrease unspecific binding treatment with blocking buffer I or
blocking buffer II is necessary (see Note 2). Blocking should be carried out for at
least 2 h at room temperature. Treatment overnight at room temperature or over
the weekend at 4 ◦ C is also possible.
3. Incubation with protein sample: Wash once with T-TBS or T-PBS for at least 5
min. After washing, incubate the membrane with the protein sample for at least 2
hat37 ◦ C. Treatment can also be carried out overnight at room temperature.
4. Incubation with a target protein binding protein (e.g., primary antibody): Wash
three times with T-TBS or T-PBS for at least 5 min each. After washing, incubate
the membrane with the (HRP-labeled) primary antibody solution for 2 h at room
temperature or 37 ◦ C.
5. Incubation with secondary antibody directed against the protein used in step 4
(if no HRP-labeled primary antibody was used): Wash three times with T-TBS or
T-PBS for at least 5 min each. After washing, incubate the membrane with the
HRP-labeled secondary antibody solution for at least 1 h at room temperature or
37 ◦ C.
6. Detection: Wash at least three times for at least 5 min with TBS or PBS. Preparation
of the chemiluminescence solution and detection (see Note 14): 22 �L of
80 mM p-coumaric acid in DMF and 50 �L of 250 mM luminol in DMF should
be mixed with 1 mL 1 M Tris-HCl pH 8.5. Add to this mixture the corresponding
amount of water (see Materials). The staining solution has to be activated by
adding 3 �L of 30% H2O2 shortly before use. Excess washing buffer on the
membrane has to be removed by placing the membrane on paper towels (see
Note 15). Use forceps for all manipulations of the membrane. Never use fingers!
The membrane then has to be placed on a plastic sheet (PP or PE). The detection
buffer is poured over the whole membrane. To ensure homogeneous distribution
the membrane has to be gently shaken on the plastic sheet. A reaction time of
approximately 1 min is recommended. Detection of the chemiluminescence can
be carried out using X-ray film or a chemiluminescence imager (62).