Synthesis and Probing of Membrane-bound Peptide Arrays-2

Incubate each membrane in 20 ml of acetylation mix for a minimum of 30 min (until all remaining blue color has disappeared).
 

Wash the membranes in 20 ml of DMF (3 times for 2 min), and then in 20 ml of alcohol (2 times for 5 min).
 

Transfer the membranes to 3MM paper folders and dry them using cold air from a hair dryer. 

Prepare 40 ml of deprotection mix.
 

Place the dried membrane in the reaction trough, add deprotection mix, close the trough very tightly, and incubate overnight with gentle agitation. This harsh treatment is required for complete cleavage of protecting groups (Kramer et al. 1999; Zander 2004). Cellulose membranes less resistant than AC-S will not survive this treatment!) 
 

Subject each membrane to the following series of washes: 
 
20 ml of DCM (4 times for 5 min each)
20 ml of DMF (3 times for 5 min each)
20 ml of alcohol (3 times for 5 min each)
20 ml of acetic acid (1 M in water) (3 times for 5 min each)
 
This is to remove the Boc group from tryptophan residues.
 
Wash each membrane with 20 ml of alcohol (3 times).

 

Transfer the membranes to 3MM paper folders and dry them using cold air from a hair dryer. Store dried membranes in a sealed plastic bag at -20°C, or process further as described in the next section.
 

Place a single membrane in a polystyrene plate, and wet it with a few drops of methanol or ethanol. 
 
This is to enhance rehydration of any peptide spots that might be hydrophobic. The peptide locations should not be visible as white spots! If this happens, extend the alcohol treatment in a sonication bath at room temperature until spots have disappeared 
 

Wash the membrane in 10 ml of TBS (3 times for 10 min each).
 

Incubate overnight in 10 ml of MBS. 
 
The blocking conditions can be critical to the success of an experiment, and, depending on the protein of interest, it may be necessary to try a number of different blocking solutions to optimize the signal-to-noise ratio. The following solutions represent increasingly stringent blocking conditions: (i) 2% (w/v) skim milk powder in TBS, (ii) 2% (w/v) skim milk powder, 0.2% (v/v) Tween-20 in TBS, (iii) MBS, (iv) MBS with 50% (v/v) horse serum. In our hands, blocking solution iii works best for most applications. 
 

Wash the membrane once in 10 ml of T-TBS for 10 min
 

Incubate for 2-4 hr in the presence of probe antibody (or protein) diluted in 8-10 ml of MBS.
 
For monoclonal antibodies, or pure proteins, use ~4-5 µg of purified antibody per milliliter of incubation volume. When using a polyclonal serum, we recommend a dilution of 1:100. It is not necessary to use a large volume of protein solution for the incubation. However, make sure that the membrane is completely covered throughout the incubation. To prevent drying out, use a lid, or seal the membrane in a plastic bag. 
 

Wash the membrane 3 times in 10 ml of T-TBS (for 10 min each).
 

Incubate for 1-2 hr with AP-conjugated secondary antibody, diluted in 10 ml of MBS.
 

Wash the membrane 2 times in 10 ml of T-TBS (for 10 min each).
 

Wash the membrane 2 times in 10 ml of CBS (for 10 min each).
 

Transfer the membrane to a flat glass tray and add 10 ml of CDS. Incubate without agitation until good signals are obtained. 
 
For individual peptides on spots, this usually takes 10-30 min; peptide pools may require longer incubations (2 hr to overnight). 
 

Stop the reaction by washing the membrane twice in PBS. Keep the membrane wet, either in PBS or covered in plastic wrap. Store at 4°C. 
 
The picture of signals on the membrane can now be documented by photography or (better) be electronically digitized with a scanner. A high-quality electronic image can be used to quantify signal intensities. Avoid drying of the membrane at this stage. If the membrane dries out, proteins may denature and become difficult to remove. After successful documentation of signals by photography or electronic scanning, continue with membrane stripping (see below, after Method B). 
 

Briefly equilibrate the peptide membrane and a sheet of nitrocellulose, trimmed to fit the peptide membrane, in transfer buffer.
 

Electro-transfer the proteins bound to the peptide spot membranes onto the nitrocellulose membrane for 1 hr at 0.85 mA/cm2. IMPORTANT: Due to SDS denaturation, all proteins will be negatively charged. Therefore, the nitrocellulose should be placed toward the positive electrode. 
 
Depending on the chemical properties of the protein ligands, the time required for the transfer might differ and, therefore, must be determined empirically. 
 

Block the nitrocellulose membrane with MBS for 2 hr at room temperature.
 

Incubate the nitrocellulose membrane for 75 min with an AP- or HRP-conjugated detection antibody, or AP-/HRP-streptavidin for biotinylated proteins diluted in MBS. 
 
Use dilutions comparable to those employed in immunoblots after SDS-PAGE. 
 

Wash the nitrocellulose membranes 3 times for 5-10 min each in T-TBS, and then 3 times for 5-10 min in TBS for 5-10 min each.
 

Remove excess buffer from the nitrocellulose membrane by gently placing tissue onto it. 
 
To avoid damage to the adsorbed protein, do not wipe or press tissue onto the membrane.

 

Detect the spots using a chemiluminescence detection kit (e.g., ECL western blotting detection reagents from Amersham Biosciences) according to the manufacturer's instructions. Be sure to include a positive control for the kit.
 
If no signal has been detected after 30 min of exposure, check that the positive control has worked, and repeat the experiment using less stringent blocking. If problems persist, this may indicate a discontinuous binding site or very low affinity binding. 
 
In case of nonspecific signals or high background, increase the stringency of the blocking conditions and make sure that the primary binding partner and detection reagent (e.g., antibody) are of high purity and are used in the highest possible dilution. 
 

Wash the spot membrane 2 times for 10 min in 20 ml of water.
 

Incubate in 20 ml of DMF, until the blue color of spot signals has dissolved (usually about 10 min; incubate in a sonication bath at 40°C if necessary). Remove the solution and wash once more for 10 min in 20 ml of DMF for 10 min. 
 
This step can be omitted if a detection method other than a dye precipitation or Method B of Stage 6 was used. 
 

Subject the membrane to the following series of washes:
 
20 ml of water (3 times for 10 min each) 
20 ml of SM-A in a sonication bath at 40°C (3 times for 10 min each)
20 ml of SM-B (3 times for 10 min each)
20 ml of alcohol (3 times for 10 min each)

 

Start the new binding assay at step 2 of the protein-binding assay protocol of Stage 6 or transfer the membranes to 3MM paper folders and dry them using cold air from a hair dryer. Store dried membranes in a sealed plastic bag at -20°C.