Blue Sepharose® 6 Fast Flow or prepacked HiTrap™ Blue HP 1 mL and 5 mL columns (Figure 4.2) can be used to remove albumin either before or after other puriﬁcation steps (Table 4.1). The albumin binds in a nonspeciﬁc manner by electrostatic and/or hydrophobic interactions with the aromatic anionic ligand, Cibacron Blue F3G-A, coupled to Sepharose®.
Figure 4.2.Prepacked with Blue Sepharose® High Performance, HiTrap™ Blue HP columns offer fast and simple removal of albumin by afﬁnity chromatography.
Use HiTrap™ Blue HP 1 mL or 5 mL columns to remove host albumin from mammalian expression systems or when the sample is known to contain high levels of albumin that might mask the UV absorption of other protein peaks.
Do not use Blue Sepharose® chromatography media if the immunoglobulin or other target molecule has a hydrophobicity similar to that of albumin.
Figure 4.3.Effective removal of albumin from human plasma using a HiTrap™ Blue HP 1 mL column.
Binding buffer: 20 mM sodium phosphate, pH 7.0, or 50 mM potassium hydrogen phosphate (KH2PO4), pH 7.0
Elution buffer: 0.02 M sodium phosphate, 2 M sodium chloride, pH 7.0, or 0.05 M potassium hydrogen phosphate, 1.5 M potassium chloride, pH 7.0
* 1 mL/min corresponds to approximately 30 drops/min when using a syringe with a 1 mL HiTrap™ column; 5 mL/min corresponds to approximately 120 drops/min when using a 5 mL HiTrap™ column.
Store in 20% ethanol at 2 °C to 8 °C.