I am trying to purify a GST tagged protein of 10kda ( with GST tag 37kda) expressed in JM109. The pI of protein is 4.08. I am using the GST beads from Sigma for affinity purification. The lysis/resuspension buffer and elution/wash buffer is PBS, pH 7.4, DTT 10mM. Elution buffer: Tris 50mM pH 8, NaCl 150mM, 10 % 0.02% NaN2, reduced glutathione 15mM. GST was found to bind to column and elute out during elutions but the protein does not bind to the column and elutes out in the flow-through. High-affinity binding beads was also tried but with no success. Any help will be of great assistance. Thank you in advance.

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