Solutions & buffers Step 1 Activation buffer. Dilute 4 μl HCl in 50 ml water for a final concentration of 1 mM Coupling buffer. Component Final concentration Stock Amount NaHCO 3, pH 8.3 100 mM 1 M 100 ml NaCl 500 mM 5 M 100 ml Add water to 1 l Step 3 Quenching buffer. Dilute 5 ml of 1 M Tris–HCl, pH 8.0 in 45 ml water for a final concentration of 100 Mm Step 4 High pH wash buffer. Component Final concentration Stock Amount Tris–HCl, pH 8.0 100 mM 1 M 25 ml NaCl 500 mM 5 M 25 ml Add water to 250 ml Low pH wash buffer. Component Final concentration Stock Amount NaOAc, pH 4.0 100 mM 1 M 25 ml NaCl 500 mM 5 M 25 ml Add water to 250 ml Storage buffer (PBS, pH 7.4) Component Final concentration Stock Amount NaCl 137 mM 5 M 1.37 ml KCl 2.7 mM 4 M 33.8 μl NaH 2PO 4 10 mM 0.5 M 1 ml K 2HPO 4 2 mM 0.5 M 0.2 ml Add water to 50 ml.

NHS-Activated Sepharose 4 Fast Flow NHS-activated Sepharose 4 Fast Flow is a pre-activated agarose matrix that increases the choice of coupling chemistries available. Suitable for coupling amino-containing smaller proteins and peptides. NHS-Activated Agarose. NHS-Activated Agarose consists of 4% cross-linked agarose that has been activated by the addition of a reactive NHS (N-hydroxysuccinimide) group. The NHS group forms covalent, chemically stable amide bonds with ligands that contain primary amines. The NHS-Activated Agarose also contains a spacer arm between.