**10. Isolation and purification of receptor for SIF isolated from** *E. coli*

The spermatozoa were washed twice with PBS (50 mM, pH 7.2) and then resuspended in same buffer. The washed spermatozoa were treated with different concentrations of NaCl i.e.1, 2, 3 and 4 M for different time intervals in order to optimize the best concentration and time combination for receptor isolation. These were incubated at 37°C under shaking conditions (150 rpm). The salt treated sample was centrifuged at 1500 g for 15 min. Cell debris was suspended in minimum amount of PBS. Both the cell debris and supernatant after dialysis and concentration (against PEG 6000) were checked for blocking of immobilization induced by SIF. Preliminary results showed that SIF receptor could be efficiently extracted by 2 M NaCl from spermatozoa when incubated for 20 h at 37°C under shaking conditions.

Purification of the receptor was further done by filtration through a Sephadex G-200 column (1.5 cm X 31 cm) equilibrated and eluted with PBS. The head pressure was maintained to achieve a flow rate of 35 ml/h. Fractions of 3 ml each were collected and each fraction was read at 280 nm (U.V. spectrophotometer). The column chromatographic pattern showed that the receptor could be eluted in the fractions 3-4 which could block the SIF induced sperm immobilization (Figure 8a). These fractions were pooled and concentrated with polyethylene glycol (PEG 6000) at 4°C and were applied to ion exchange column. First of all, 80 ml of elution buffer (PBS) was allowed to run down the column. Final elution was done with 0.05, 0.1, 0.2, 0.4 and 0.6 M NaCl dissolved in PBS. Fractions of 4 ml each were collected and read at 280 nm on U.V. spectrophotometer. The fractions showing blockage of sperm immobilization i.e. 2-4 were pooled and concentrated (Figure 8b). Molecular weight of the purified SIF receptor was estimated by SDS-PAGE. The purified SIF receptor was first denatured and then loaded onto the gel along with the standard protein markers. After the gel was run, silver staining was done and molecular weight was estimated to be ~113 kDa (Figure 8c) [25].

306 Chromatography – The Most Versatile Method of Chemical Analysis

7b) [24].

cm) equilibrated and eluted with PBS. The head pressure was maintained to achieve a flow rate of 35 ml/h. Fractions of 3 ml each were collected and each fraction was read at 280 nm. Fractions showing blockage of agglutination activity were pooled and concentrated with PEG 6000 at 4°C (Figure 7a). The homogeneity of the preparations was checked by PAGE and the molecular weight of the purified receptor was estimated by SDS-PAGE. The molecular weight of the receptor for SAF from *E. coli* was approximately, 125 kDa (Figure

7a 7b

spermatozoa when incubated for 20 h at 37°C under shaking conditions.

**Figure 7. (a)** Elution pattern of receptor from human sperm on Sephadex G-200 column. **(b)** SDS-PAGE (molecular weight determination) **Lane 1**: Standard proteins markers, **Lane 2**: Purified receptor protein

The spermatozoa were washed twice with PBS (50 mM, pH 7.2) and then resuspended in same buffer. The washed spermatozoa were treated with different concentrations of NaCl i.e.1, 2, 3 and 4 M for different time intervals in order to optimize the best concentration and time combination for receptor isolation. These were incubated at 37°C under shaking conditions (150 rpm). The salt treated sample was centrifuged at 1500 g for 15 min. Cell debris was suspended in minimum amount of PBS. Both the cell debris and supernatant after dialysis and concentration (against PEG 6000) were checked for blocking of immobilization induced by SIF. Preliminary results showed that SIF receptor could be efficiently extracted by 2 M NaCl from

Purification of the receptor was further done by filtration through a Sephadex G-200 column (1.5 cm X 31 cm) equilibrated and eluted with PBS. The head pressure was maintained to achieve a flow rate of 35 ml/h. Fractions of 3 ml each were collected and each fraction was read at 280 nm (U.V. spectrophotometer). The column chromatographic pattern showed that the receptor could be eluted in the fractions 3-4 which could block the SIF induced sperm immobilization (Figure 8a). These fractions were pooled and concentrated with polyethylene glycol (PEG 6000) at 4°C and were applied to ion exchange column. First of all, 80 ml of elution buffer (PBS) was allowed to run down the column. Final elution was done with 0.05, 0.1, 0.2, 0.4 and 0.6 M NaCl dissolved in PBS. Fractions of 4 ml each were collected

**10. Isolation and purification of receptor for SIF isolated from** *E. coli* 

**Figure 8. (a)** Elution pattern of receptor from human sperm on Sephadex G-200 column. **(b)** Elution pattern of receptor from human sperm on DEAE cellulose column. **(c)** SDS-PAGE (molecular weight determination) **Lane 1**: Standard proteins markers, **Lane 2**: Purified receptor protein
