Low molecular weight markers

To evaluate and visualize the effect of TFA depletion of the most abundant proteins from this snail hemolymph, 1D SDS-PAGE were carried out on 12% gel at constant 20 mA in a Miniprotean 2D-chamber (BioRad). Supernatants obtained after depletion were mixed in
a ratio of 1: 1 with loading buffer and under reducing conditions were heated at 95°C for 5 min prior to loading onto the gels. Briefly, 1st lane was loaded with 3.76 mg/ml of non-depleted hemolymph, 2nd lane was loaded with 2.32mg/ml of 0.75% TFA depleted
hemolymph and 3rd lane was loaded with 2.03 mg/ml of 0.5% TFA depleted hemolymph. To facilitate comparisons, all samples were run simultaneously with low molecular weight markers (BioRad). After electrophoretic separation, the silver stained [10 ]
(Shevchenko et al., 2000). The imaged of the gel was scanned on Image Scanner III (GE Healthcare) and exported using the labscan tools version 6.01. This analysis intended the evaluation of (1) the number of bands in each depletion compared to the non-depleted
hemolymph and (2) to compare the huge bands at the beginning of the gel.

SDS-PAGE was performed as previously described [24 (link)] using low-molecular-weight markers (2000–250,000 Da, Bio Rad, Hercules, CA, USA). The electrophoresis was performed for 50 min at 120 mV, and the gels were stained following the Coomassie method [25 ].