**6. Extraction and purification of SIF from** *E. coli*

The isolate of *E. coli* showing immobilization of spermatozoa was grown in BHI broth under shaking conditions (150 rpm) at 37ºC for 72 h. The culture was centrifuged at 10,000 g for 15 min at 4ºC and cell-free supernatant was prepared by passing the supernatant through a 0.22 µm Millipore filter. The supernatant was then subjected to ammonium sulphate precipitation so as to get 20, 40, 60, 80, and 100% saturation. The precipitates so obtained were dissolved in a minimum amount of PBS. The precipitated protein was dialyzed against PBS under cold conditions and checked for sperm immobilization. SIF could be saturated at 60-80% of ammonium sulphate. Further purification of the factor was done by gel filtration through a Sephadex G-100 column (2 cm x 31 cm) equilibrated, and eluted with PBS.

Isolation and Purification of Sperm Immobilizing/Agglutinating Factors from Bacteria and Their Corresponding Receptors from Human Spermatozoa 303

To verify the purification status of all the preparations, PAGE was carried out (10% resolving and 5% stacking gel). Estimation of molecular weight by SDS-PAGE, showed that SIF had a molecular weight of ~56 kDa (Figure 4c), compared to standard protein

4a

4b

4c **Figure 4. (a**) Elution pattern of SIF from *E. coli* on Sephadex G-100 column. **(b)** Elution pattern of SIF from *E. coli* on DEAE cellulose column. **(c)** SDS-PAGE of purified SIF, with **Lane1** containing Standard protein marker and **Lane2** containing DEAE cellulose purified and concentrated fraction. Molecular

markers [21].

weight approximately, 56 kDa.

**Figure 3. (a)** Elution pattern of SAF obtained after Sephadex G-200 gel filtration of dialyzed and filtered supernatant containing sperm ligand on *E. coli***,** showing the presence of ligand in fractions 6-9 with peak value in fraction 7 (arrow refers to sperm ligand on *E. coli*). **(b)** DEAE cellulose chromatography of G-200 pooled and PEG concentrated fractions revealed the presence of three peaks but the agglutinating activity was present only in the fractions 46-49 with peak value in fraction 48 (arrow refers to sperm ligand on *E. coli*). **(c)** SDS-PAGE of purified sperm ligand, with **Lane1** containing Standard protein marker and **Lane2** containing DEAE cellulose purified and concentrated fraction. Molecular weight approximately, 71 kDa.

Fractions of 3 ml each were collected and each fraction was read at 280 nm on U.V. spectrophotometer. Fractions 8-14, showing the immobilization of spermatozoa were pooled and concentrated using PEG 6000 under cold conditions (Figure 4a). These fractions were applied on to a DEAE cellulose column. First of all, 80 ml of elution buffer, PBS (50 mM pH 7.2) 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 (50 mM, pH 7.2). Fractions of 4 ml each were collected and read at 280 nm on U.V. spectrophotometer. The fractions showing immobilization of spermatozoa i.e. 3-7 were again pooled and concentrated (Figure 4b). To verify the purification status of all the preparations, PAGE was carried out (10% resolving and 5% stacking gel). Estimation of molecular weight by SDS-PAGE, showed that SIF had a molecular weight of ~56 kDa (Figure 4c), compared to standard protein markers [21].

302 Chromatography – The Most Versatile Method of Chemical Analysis

approximately, 71 kDa.

3c **Figure 3. (a)** Elution pattern of SAF obtained after Sephadex G-200 gel filtration of dialyzed and filtered supernatant containing sperm ligand on *E. coli***,** showing the presence of ligand in fractions 6-9 with peak value in fraction 7 (arrow refers to sperm ligand on *E. coli*). **(b)** DEAE cellulose chromatography of G-200 pooled and PEG concentrated fractions revealed the presence of three peaks but the agglutinating activity was present only in the fractions 46-49 with peak value in fraction 48 (arrow refers to sperm ligand on *E. coli*). **(c)** SDS-PAGE of purified sperm ligand, with **Lane1** containing Standard protein marker and **Lane2** containing DEAE cellulose purified and concentrated fraction. Molecular weight

Fractions of 3 ml each were collected and each fraction was read at 280 nm on U.V. spectrophotometer. Fractions 8-14, showing the immobilization of spermatozoa were pooled and concentrated using PEG 6000 under cold conditions (Figure 4a). These fractions were applied on to a DEAE cellulose column. First of all, 80 ml of elution buffer, PBS (50 mM pH 7.2) 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 (50 mM, pH 7.2). Fractions of 4 ml each were collected and read at 280 nm on U.V. spectrophotometer. The fractions showing immobilization of spermatozoa i.e. 3-7 were again pooled and concentrated (Figure 4b).

3a 3b

**Figure 4. (a**) Elution pattern of SIF from *E. coli* on Sephadex G-100 column. **(b)** Elution pattern of SIF from *E. coli* on DEAE cellulose column. **(c)** SDS-PAGE of purified SIF, with **Lane1** containing Standard protein marker and **Lane2** containing DEAE cellulose purified and concentrated fraction. Molecular weight approximately, 56 kDa.
