His-Trple A protein construct into DE3 cell, pellet were suspended in 10mM HEPES ph 7.4 250mMkcl and 10%glycerol, The resulting pellet resuspending in 100mM Tris ph7.0,0.5M=mM EDTA,5mMDTT,2 M urea, TritonX-100,
Then treated with extraction buffer (10mM HEPES,250mMkcl,6M guanidine HCl but after chromatography (NI-NTA affinity) protein aggregated, what should i do
Does the target protein have Cys-Cys ? These may need reshuffling with dialysis in a mixture of cysteine cystine. If no C-C bonds expected maybe try dialysis out of the GuHCl buffer into NDSB-201 (Non-detergent sulfobetaine). Good luck!
Edward Michelini , Yes protein has cys-cys. Protein sequence is 45 amino acid sequence. Nine cys residue present within this sequence. If the aggregation is due to cys-cys, what should i do?
Protein aggregation can result from various factors, including improper refolding, buffer composition, suboptimal pH, and insufficient stabilization conditions. In your case, the likely causes and solutions are:
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