His GraviTrap™ TALON® columns are prepacked with 1 ml of TALON® Superﬂow medium. Each column provides simple manual puriﬁcation of up to 15 mg of histidine-tagged proteins. The different puriﬁcation steps are performed using gravity ﬂow and require no further equipment (Figure 1). Large sample volumes can be applied in one go, and the histidine-tagged protein is effectively eluted in a small volume. Each package contains 10 prepacked columns, and each column is delivered in a package that converts into a column stand (Workmate). The plastic tray in the product package can be used to collect liquid waste. The columns are manufactured from biocompatible polypropylene, and special frits in each column protect the medium from running dry during puriﬁcation. Visit Characteristics of Ni Sepharose, Ni Sepharose excel, TALON® Superflow, and uncharged IMAC Sepharose products.
Figure 1a.His GraviTrap™ TALON® column is a prepacked gravity-ﬂow column for convenient manual puriﬁcation of histidine-tagged proteins.
Figure 1b.Schematic workﬂow for purifying histidine-tagged proteins on His GraviTrap™ TALON® column.
Refer to Puriﬁcation using TALON® Superﬂow earlier in this chapter for a general procedure for sample and buffer preparation.
The protein binding capacity of the column is approximately 15 mg of histidine-tagged protein/ column (protein dependent). Connecting LabMate reservoir (see Ordering information) to the column increases convenience when handling volumes above 10 ml. This raises the loading capacity to approximately 35 ml in one run.
After thorough cell disruption, it is possible to apply the unclariﬁed sample directly to the column without experiencing clogging problems.
If using buffers that contain denaturing agents or viscous solutions, perform the puriﬁcation at room temperature.
His GraviTrap™ TALON® column enables fast and easy manual puriﬁcation of histidine-tagged proteins without the need for a puriﬁcation system. Using a gravity-ﬂow protocol, histidine-tagged green ﬂuorescent protein (GFP-His) added to E. coli lysate was puriﬁed with His GraviTrap™ TALON® columns in less than 30 min. The recovery was calculated using absorbance measurements and was found to be 95%. Figure 3.43 shows the SDS-PAGE analysis of the ﬁrst and second elution fractions. The results indicate the purity of ≥ 93% for the puriﬁed GFP-His protein.
Figure 2. SDS-PAGE analysis of elution fractions from a puriﬁcation of GFP-His (Mr 28 000) added to E. coli lysate. The SDS-polyacrylamide gel (reducing conditions) was analyzed with ImageQuant TL software. The experiment was performed in triplicate. The ﬁrst elution fraction contained 96% of the puriﬁed protein.