We perform solubilization of protein in 8M urea and 100mM cysteine and refold it in tris buffer pH 8.2 by dilution method.then we adjust its pH to 4.5. we get good results until then as seen from HPLC. but when we load this sample on sp sepharose fast flow column and elute it using na acetate 25mM buffer + 0.4 M NaCl ph 4.5 we do not get any elution. and on washing of column with NaOH in reverse direction some peak is observed. why is it so. why do we not get out protein eluted in naCl. how do we go about this. initially we used to perform solubilization using 8M urea only no cysteine and used CuSO4 fo air oxidation in refolding. this when we load we get protein elution. but the modified method does not give any whereas the yield of solubilization refodlign is almost double by modified method. how can we solve this.