Protein Purification

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vvsgrat
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Protein Purification

Hai,
I am facing problem in protein purification. Mine is a peptide of Molecular weight ~3 kDa and is cationic in nature (and most peptide bacteriocins are cationic). I use S-Ceramic Hyper D strong cationic column (Pall Scientific) with 10mM citrate buffet pH 4.0. Protein concentration 50mg/ml (loading capacity of the column is 75mg/ml) But my protein is getting bound partly only. Major is geting unbound (nearly 80-90%). I also tried with pH of 5.0 acetate buffer but is the same case. I also diluted the sample much with staring buffer (2X also) to bring the ionic strength of sample nearly to the buffer but it does not solve. How to go about for geting maximum binding.
In case of elutin (Gradient 0-1M) it elutes as a single peak with 0.1 to 1M (Broad peak). I even altered the flow rate but still its like same. Please help me for purification using IEX.