Colloidal coomassie

Immuprecipitations were performed as previously described [18 (link)] using anti-FLAG, anti-HA agarose or anti-myc agarose (Sigma) and eluted using Flag peptide (150μg /ml) or HA peptide (100μg /ml) (Sigma). For mass spectrometry studies, the eluted samples were subjected to SDS-polyacrylamide gel, stained by colloidal Coomassie (Bio-Rad). Prominent bands were cut out, digested with trypsin overnight and analyzed by mass spectrometry as described before [18 (link)]. The acquired peptide masses were interrogated by ProFound, a protein identification database. For direct interaction studies proteins were produced by in vitro transcription/translation kit (Promega) following the manufacturer's protocol and mixed in the presence of the appropriate agarose beads.

The protein composition of mucus was first analyzed by one-dimensional electrophoresis (1-DE). Initially, mucus samples from all animals (CTRL and M-ST fish) were pooled, and triplicate samples (54-56 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a TGX Any kD precast gel (Bio-Rad, Hercules, CA, USA) run at 200V for 25 min and stained overnight with colloidal Coomassie (Bio-Rad). The gel was then divided into 10 slices (0.65 cm) that were analyzed independently. Proteins in the gel were digested with protein-grade trypsin (Promega, Madison, WI, USA) and concentrated by speed vacuum at a final volume of 12 μL for mass spectrometry.

Labeled samples (250 µL) were applied to IPG strips (13 cm, pI ranges 3–10, GE-Healthcare) on the rehydration tray (GE-Healthcare) and focused using an Ettan IPGphor II (GE-Healthcare) as follows: active rehydration at 30 V for 14 h, followed by isoelectric focusing for a total of 28 kV/h (step to 500 V for 1 h, step to 1000 V for 1 h, step to 8000 V to a total of 28 kV/h). After isoelectric focusing, disulfide bonds were reduced by placing the strips for 10 min in 20 mL equilibration buffer (6 M urea, 50 mM Tris, pH 8.8, 30% glycerol, 2% SDS) containing 5 mg / mL DTT. The strips were then incubated for 10 min in fresh equilibration buffer with 45 mg / mL iodoacetamide. For the second dimension, the IPG strips were placed on 12% homogeneous polyacrylamide gels (4% stacking). Gels were cast using low-fluorescence glass plates (13 cm plates, GE-Healthcare) previously treated with bindsilane (GE-Healthcare). Each SDS-PAGE was run at 9 mA for 16 h in a HOEFER SE-600 system. Individual images of Cy2-, Cy3- and Cy5-labeled proteins of each gel were obtained using a Typhoon 9410 scanner (GE-Healthcare) with excitation / emission wavelengths of 480 / 530 nm for Cy2, 520 / 590 nm for Cy3 and 620 / 680 nm for Cy5. After imaging the gels were stained with colloidal Coomassie (Bio-Rad, Hercules, CA, USA).