2.9. Experimental Protocol
The precise step-by-step protocol used in our experimental pipeline is given bellow, capturing all important observations that lead to reproducible and good quality SPOT assays in our lab (buffer recipes are also provided at the end of the protocol):
Day 1: Blocking and hybridization of the membrane (TIMING ~9 h) 
1| Rehydrate the membrane: rinse several times with 100% EtOH then equilibrate 3 × 5 min in PBST (see below).
2| Block the membrane with 10 mL 5% BSA in 1X PBST buffer for at least 8 h at room temperature (use a rocking table).
▲ CRITICAL: Always adjust the amount of solution according to the size of the membrane (and the container used to handle it). The membrane must be covered by the solution on every step.
▲ CRITICAL: Milk can be used for blocking but gives much higher background.
3| Wash the membrane 2 × 5 min in 1X PBST buffer then 1 × 5 min in PBS buffer at room temperature.
4| Add 1 µM (final concentration) of the protein of interest diluted in 10 mL 1X PBS buffer (volume adjustable); leave overnight on a rocking table at 4 °C.
Day 2: Development and reading (TIMING ~5 h) 
5| Wash the membrane 3 × 5 min with 1X PBST buffer to remove any unbound protein.
6| Block the membrane with 10 mL of 5% BSA in 1X PBST buffer for 1 h at room temperature.
7| Wash the membrane 3 × 5 min with 1X PBST buffer.
8| Add HPR-conjugated His antibody (1:3000 dilution) in 1% BSA PBST buffer and incubate for 1 h at room temperature.
9| Wash the membrane 3 × 15-20 min each in 1X PBST buffer.
▲ CRITICAL: These washes are critical to avoid high background.
10| Place the membrane quickly on drying paper to remove any liquid excess.
▲ CRITICAL: The membrane should not be left to dry completely.
11| Develop using an ECL kit; mix an equal amount of the peroxide solution and the Luminol Enhancer solution (toxic so always wear gloves) and cover the membrane. Let the reaction develop for one minute.
12| Remove the excess of the ECL solution and read the result on a chemiluminescence compatible imaging station (we use the ImageQuant LAS-4000 camera set on chemiluminescence settings) first for 1 min to check that the reaction develops properly, then for 80 min with 5 min increments.
13| Save the resulting image data in the TIF graphic format.
Day 2: Stripping—Part I (TIMING ~3 h) 
14| Rinse the membrane 3 times in distilled water.
15| Incubate the membrane in the Restore Western Blot Stripping Buffer for 30 min at 37 °C.
16| Wash the membrane 3 × 10 min in distilled water at room temperature.
17| Incubate 2 × 45-60 min in Stripping buffer A (see below) at room temperature.
18| Leave overnight at room temperature in Stripping buffer A with 500 µL of NiNTA beads to trap the released His-tagged protein.
Day 3: Stripping—Part II (TIMING ~4 h) 
19| Incubate 2 × 30 min in Stripping buffer B (see below) at room temperature.
20| Wash 3 × 10 min in distilled water at room temperature.
21| Wash 3 × 10 min in distilled water at 60 °C.
22| Wash 1 × 10 min in distilled water at room temperature.
23| Wash 1 × 30 min in 90% TFA at room temperature.
▲ CRITICAL: TFA is a very strong corrosive acid. Always wear gloves and protective goggles and leave the membrane in a fume hood during the reaction.
24| Wash the membrane 2 × 10 min in distilled water at room temperature.
25| Re-equilibrate the membrane 3 × 10 min in 1× PBST buffer.
■ PAUSE POINT If the membrane needs to be used the following day, it can be left overnight into 1X PBST buffer.
■ PAUSE POINT If the membrane is no longer needed, it can be stored at −20 °C for several months. In this case, the membrane needs to be dehydrated.
26| Wash the membrane 2 × 10 min in 20% EtOH at room temperature.
27| Wash the membrane 2 × 10 min in 50% EtOH at room temperature.
28| Wash the membrane 2 × 10 min in 95% EtOH at room temperature.
29| Let the membrane dry at room temperature overnight, wrap it in aluminum foil and store at −20 °C in a zip bag.
Day 4: Quality control of the stripping (TIMING ~5 h) 
30| Repeat steps 6 to 13.
31| If the membrane has been properly stripped (no signal apart from the positive controls), repeat steps 14 to 16, then go to step 25 or 26.
32| If the membrane still displays SPOTs in addition to the positive controls, the stripping process can be repeated (steps 14 to 30), but it is possible that a new membrane will have to be re-synthesized.
Solution/buffer recipes 
PBS Buffer (20×, 2L): Prepare by mixing 320 g of NaCl, 8 g of KCl, 57.6 g of Na2HPO4, 9.6 g of KH2PO4 in deionized water for a total volume of 2000 mL. Adjust pH to 7.4 with HCl.
PBST Buffer (20×, 2L): Prepare by mixing 320 g of NaCl, 8 g of KCl, 57.6 g of Na2HPO4, 9.6 g of KH2PO4, 20 mL of Tween-20 in diionized water for a total volume of 2000 mL. Adjust pH to 7.4 with HCl.
Stripping buffer A (1×, 1L): Prepare by mixing 573.18 g of guanidinium HCl salt (6M final concentration) and 10 mL Triton X-100 (1% final concentration) in distilled water for a total volume of 1000 mL.
Stripping buffer B (1×, 1L): Prepare by mixing 34.38 g of imidazole (500 mM final concentration), 29.22 g of NaCl (500 mM final concentration) and 20 mL of 1 M TRIS.HCl pH 7.5 (20 mM final concentration) in distilled water for a total volume of 1000 mL.
Materials needed His-tag® Antibody HPR conjugated, Novagen, distributed by Merck-Millipore, #71841.Pierce® ECL Western blotting Substrate, Thermo Scientific distributed by Fisher Scientific, #32106Bovine Serum Albumin Heat Shock Reagent grade powder pH7.0, Fisher Chemical, BPE 1600-100.Restore Western Blot Stripping Buffer, Thermo Scientific, #21059.