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Be sure that all the purification buffers are at 4 °C and carry out the protein purification in the cold room to keep away from protein denaturation. BE AWARE: This avoids the coupling of amino acids within the additional cycles with the membrane instead of the [rising peptide](https://latanyakeith.com/author/mitzisommers14/) chain. NOTICE: After the coupling of the first C-terminal amino acid of all peptides to the amino group of the membrane, the amino teams between the spots and also some of the amino teams within the spot areas don't kind bonds with the amino acids.
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