?(Fig.3)3) were subjected to the same experiments as described above. situation explained for additional EP proteins 9, 10, 18, MSP22.8 constitutes the first individual EP protein that is also found while a shell matrix protein. In the work explained here, we carried out a multistep purification strategy to isolate and determine MSP22.8 from EPF. A sequencing\aided database search was used together with a classical database search and it was concluded that MSP22.8 is a protease inhibitor\like protein that has a strong similarity with A0A0K0YAZ2 (protease inhibitor\like protein\B1), which was previously described for and and in the shell organic matrix of and EPF was subjected to ammonium sulfate (AS) precipitation, MSP22.8 was detected mainly in fractions corresponding to 50% and 75% AS (Fig. ?(Fig.11). Open in a separate window Number 1 Western blot of EPF fractions acquired by AS precipitation. (1) Pellet acquired at 75% AS; (2) pellet acquired at 25% AS; (3) pellet acquired at 50% AS; (4) supernatant at 75% AS; (5) molecular mass markers; (6) EPF control. The top band of MSP22.8 (100 kDa) was observed in all fractions, even though pellet acquired at 50% AS showed the strongest positive reaction (Fig. ?(Fig.1,1, Collection 3). The lower band of MSP22.8 (approximately 55 kDa) was only detected in the EPF precipitated with 75% AS. Bands related to a molecular mass above 130 kDa were detected simultaneously in pellets acquired at 25% and 50% AS, which suggests the presence of multimers or aggregates of MSP22.8. Bands were not detected in the remaining supernatant after precipitation with 75% AS. Immunoaffinity chromatography Given that the two intense bands of interest observed in the EPF portion (the top and the lower Impulsin bands) were only present in the EPF portion at 75% AS, this Impulsin Rabbit polyclonal to ABHD12B portion was further purified using an affinity chromatography column. The column was prepared with mAb M22.8 in order to capture specifically the antigen MSP22.8. The eluted fractions were then evaluated by western blot (Fig. ?(Fig.22). Open in a separate window Number 2 Immunoaffinity chromatography of the EPF portion acquired at 75% AS. Colorimetric western blot after 10% SDS/PAGE (under reducing conditions). (1) EPF before moving through the column; (2) concentrated eluted portion after immunoaffinity chromatography using mAb M22.8 in the column; (3) EPF wash; (4) M22.8 hybridoma supernatant; (5) molecular mass markers. It can be seen in Fig. ?Fig.22 that strong bands at 55 and 100 kDa look like concentrated in the purified portion (lane 2), indicating that the mAb is able to capture both bands. Bands (called IC bands) from different experiments were by hand excised from gels for further analysis by mass spectrometry. Protein purification by fast protein liquid chromatography (FPLC) Impulsin Pellets acquired at 75% AS were also processed by FPLC. Eluates were checked by dot blot to confirm the presence of MSP22.8 (data not shown). Only those samples that tested positive were assayed by western blot. It can be seen in Fig. ?Fig.33 that MSP22.8 was detected in two peaks, namely 3 and 4. Open in a separate window Number 3 Chromatogram and western blot of EPF fractions acquired at 75% AS. Five microlitre of EPF fractions related to peak figures 3 and 4 was assayed by western blot after SDS/PAGE (10%) under reducing conditions. (mAU) milli absorption devices. In peak number 3 3, the 1st 11 fractions only showed the band at 100 kDa. However, a band at 70 kDa started to appear in the transitional zone between peaks 3 and 4 (Fig. ?(Fig.3,3, western blot of fractions of maximum number 3 3). Peak number 4 4 showed two bands (around 55 and 70 kDa) in the 1st seven fractions, and thereafter, only the 55 kDa was band was visible in.