Preparation and purification of different chemicals used are as follows and all other reagents used were of analytical grade without further purification. Purification of sHSPs ; Overproduction of sHSPs by E. Coli cells and purification of the overproduced sHSPs were done as described by Hossain and Aso (2010). Thus N-terminal His-tagged recombinant sHSPs were prepared. Protein amounts for sHSP19.9 and sHSP20.8 were calculated using molar extinction coefficients (ε280); 19940 cm-1 M-1 and 22585 cm-1 M-1, (Pace et al., 1995). Buffer used ;Unless otherwise noted, the used buffers were 50 mM sodium phosphate (pH 8.0) containing 0.1 M NaCl (buffer A), containing 0.3 M NaCl (buffer B), and 20 mM HEPES (pH 7.7) containing 10 mM NaCl (buffer C; low ionic strength buffer).Preparation of BLC solution and assay of BLC activity; BLC was purchased from Sigma (Tokyo, Japan). BLC solution was freshly prepared with the above mentioned buffers where necessary and used without further purification. Concentration of the BLC was determined by spectrophotometry, using ε280 = 93.706 mM-1cm-1 (Decker, 1977). Thermal aggregation assay of BLC; Concentration-, time- and ionic strength- dependent thermal aggregation of BLC was monitored at 60ºC by spectrophotometer (Shimadzu UV-2400) in the presence of buffer C. For the first one, different concentration of BLC ranged from 5.20 to 13.0 µM was used, for the second one, 5.0 μM BLC was used to monitor the aggregation up to 30 min with 5 min intervals and for the last one, 5.0 μM BLC with different NaCl concentration (ionic strength); 0.01 M, 0.1 M and 0.3 M containing 20 mM HEPES was used and the mode of aggregation was studied at 360 nm. CLA of sHSP19.9 and sHSP20.8 in the presence of high ionic strength buffers: sHSP19.9 was thermally aggregated at 60ºC in the presence of buffer C, which was suppressed in the presence of high ionic strength buffers, its self aggregation was suppressed (Hossain et al., 2010). So, high NaCl concentration in the buffer, the CLA of sHSP19.9 against BLC aggregation was monitored. Two different methods; 96-wells plate (Bio-Rad) and spectrophotometer were used to observe the CLA of sHSP19.9 in the presence of buffer A. For 96-well plate method, the activity was observed with different concentration of sHSP19.9 such as 0 (control), 0.025, 0.05, 0.075, 0.10, 0.125, 0.150, 0.175 and 0.20 mg/ml against fixed concentration (5.0 mg/ml) of BLC. Absorbance was monitored by microplate reader (Bio-Rad) at 595 nm. For spectrophotometer, the activity was observed at 415 nm for 20 min with a series of molar concentration ratio such as 1:0 (control; BLC 5.0 µM only), 1:0.1, 1:0.2, 1:0.5, 1:1 and 1:2. Molar concentration ratio-dependent CLA of sHSP19.9 against BLC aggregation in the presence of same buffer was also monitored at 415 nm. The used molar concentration ratio was kept at 1:1 (fixed) but their concentration was different such as 5.0, 7.5 and 30.0 µM for each. Beside buffer A, other high ionic strength buffers such as 0.1 M and 0.3 M NaCl concentrations containing 20 mM HEPES buffer were also used. For 0.1 M NaCl containing 20 mM HEPES buffer (pH 7.7), 1:0 (control; BLC 5.0 µM only), 1:0.1, 1:0.2, 1:0.5, 1:1 and 1:2 molar concentration ratio was used. The molar concentration was 1:0 (control; BLC 2.5 µM only), 1:1, 1:2, 1:3, 1:4 and 1:8 for 0.3 M NaCl containing 20 mM HEPES buffer (pH 7.7). On the other hand, various molar concentrations ratio such as 1:0 (control; 5.0 µM BLC only), 1:0.1, 1:0.2, 1:0.5, 1:1 and 1:2 for 20 mM HEPES buffer (pH 7.7) containing 0.1 M NaCl, 1:0 (control; 2.5 µM BLC only), 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:1 and 1:2 for 20 mM HEPES buffer (pH 7.7) containing 0.3 M NaCl and 1:0 (control; 5.0 µM BLC only), 1:0.05, 1:0.1, 1:0.2 and 1:1 for buffer A were used for sHSP20.8. The same molar concentration and the same procedure used for sHSP19.9 were followed for sHSP20.8. Complex formation among sHSPs and BLC; Both sHSPs separately with BLC at 1:1 molar concentration ratio was incubated at 60ºC for 1 hour to observe the probable complex formed among them with high ionic strength [20 mM HEPES buffer (pH 7.7) containing 0.3 M NaCl]. The incubated sample was cooled at room temperature for 1 hour and centrifuged at 14,000 rpm for 20 minutes, and the supernatant was used to observe the existence of protein by spectrum before application to gel filtration column.