**8. Isolation and purification of receptor for SIF isolated from** *S. aureus*

304 Chromatography – The Most Versatile Method of Chemical Analysis

was estimated to be approximately, 57kDa (Figure 5b) [22].

5a 5b

**Figure 5. (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.

**SAF isolated from** *S. aureus* 

**7. Isolation and purification of receptor from human spermatozoa for** 

Spermatozoa were washed twice with PBS and resuspended in the same buffer. Washed spermatozoa were treated with 1, 2, 3 and 4 M NaCl for different intervals to optimize the best concentration and time combination for receptor extraction. The treated mixture was centrifuged at 10,000 g for 20 min. The pellet and supernatant were examined for blockage of agglutination activity. Receptor was efficiently extracted by 3 M NaCl when incubated for 12 h at 37°C while shaking at 150 rpm. Crude receptor was further dialyzed extensively against PBS under cold conditions and then concentrated using PEG 6000. Receptor was purified by filtration through a 2 x 31 cm Sephadex G-200 column, equilibrated and eluted with PBS. Head pressure was maintained to achieve a flow rate of 10 ml/h. Fractions (3 ml) were collected and each was read at 280 nm using a U.V. spectrophotometer. Fractions were analyzed for the presence of receptor by blocking the agglutination of spermatozoa by SAF. Fractions (12-17) representing receptor were pooled, concentrated against PEG 6000 at 4°C (Figure 5a). One-dimensional SDS-PAGE was done using a vertical slab gel apparatus with stacking gel containing 5% polyacrylamide and resolving gel containing 12% polyacrylamide. The protein sample was diluted 1:1 with reducing sample buffer and heated for 5 min at 100°C. The sample was loaded and subjected to electrophoresis at 50 V for 15 min, followed by 150 V for 45 min. The gel was stained with Coomassie Brilliant Blue R-250 and calibrated using molecular weight markers. The molecular weight of the receptor Salt extraction of the receptor from human spermatozoa was done by treating the washed sperm sample with 1, 2, and 3 M NaCl for different time intervals i.e. 2, 4, 8, 12, 24 and 48 h. Purification of the receptor was further done by gel filtration through a Sephadex G-100 column (2 cm × 31 cm) equilibrated and eluted with PBS (50 mM, pH 7.2). Fractions of 3 ml each were collected and absorbance read at 280 nm to determine the SIF receptor concentration. Fractions showing the blockage of immobilization of spermatozoa (2-5, with a peak in fraction 3) were pooled and concentrated using PEG 6000 under cold conditions (Figure 6a). To check the purification status and biological activity of pooled and concentrated fractions, PAGE was carried out. Molecular weight of the purified receptor as estimated using SDS-PAGE was found to be approximately, 62kDa (Figure 6b) [23].

**Figure 6. (a)** Elution pattern of receptor from human sperm on Sephadex G-100 column. **(b)** SDS-PAGE (molecular weight determination) **Lane 1**: Standard proteins markers, **Lane 2**: Purified receptor protein

## **9. Isolation and purification of receptor for SAF isolated from** *E. coli*

The corresponding receptor for SAF was isolated from human spermatozoa using the ligand as a tool. The procedure involved the use of salt solution for the extraction of molecules bound to the cell surface. The spermatozoa were washed twice with PBS and then resuspended in same buffer. They were then treated with different concentrations of NaCl i.e. 1, 2, 3 and 4 M for different time intervals and 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 cell debris and supernatant were dialyzed against PBS at 4°C overnight, concentrated against PEG 6000 and checked for blocking of agglutination induced by SAF. Results showed that receptor for SAF from *E. coli*  could be efficiently extracted by 2 M NaCl when incubated for 18 h. Purification of the receptor was further carried out 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. 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 7b) [24].

Isolation and Purification of Sperm Immobilizing/Agglutinating Factors from Bacteria and Their Corresponding Receptors from Human Spermatozoa 307

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

8a

8b

8c **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

(Figure 8c) [25].

**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
