Description

The i CapTag ^TM resin is designed to capture recombinant proteins tagged with the i CapTag ^TM , a self-cleaving affinity tag for the rapid and simple purification of tagless proteins.

Prepacked i CapTag ^TM columns are designed for simple purification of tagless proteins via a self-cleaving affinity tag.

• The i CapTag ^TM resin binds with high specificity to the i CapTag ^TM , a 36-amino acid sequence appended to the N-terminus of the target protein.
• Strong and selective binding at pH 8.5 allows purification of the target protein via a conventional affinity purification approach, where cleaving of the tag is minimized at alkaline pH.
• Once purified, the i CapTag ^TM is induced to release the target protein from the bound tag via a change in buffer pH from 8.5 to 6.2.
• Controllable cleavage to 90% released target typically takes place in less than 5 hours at room temperature (depending on the N-terminal amino acids of the target protein). Prescribed modifications of the first two amino acids of the target can be used to optimize cleaving. In some cases, cleaving can take place in minutes.
• Once the released target protein is recovered in the column effluent, the i CapTag ^TM resin can be stripped at reused by a low pH wash.
• The i CapTag ^TM resin has been used successfully for proteins expressed in E. coli , CHO and HEK cell lines, as well as cell-free systems and non-standard host cells.

Product Specifications

Table 1. Characteristics of the i CapTag ^TM affinity chromatography.

Parameters	Requirements*
Packing size	1 x 600 µL per column (set of 8 gravity columns)
Bed dimensions	5 mm ID x 30 mm H (Internal Dimension (ID) x Height (H))
Bed volume	600 µL
Column material & column working temperature and pressure	Polypropylene, 4°C to 30°C, max. working pressure up to 8.0 bar
Flow rate**	The commonly used flow rate is between 150 - 600 µL/min. Do not exceed the flow rate of 0.98 mL/min.
Ligand Density	15 ± 2 g/L
Cleavability &	95% or higher cleavage in 5 hours at room temperature (based on GFP test target protein)
Base Bead	Hardened agarose (6% cross-linked agarose)
Particle Size, d 50v	~90 μm
Maximum Back Pressure	40 psi (2.8 bar; 0.28 MPa)
Typical Working Temperature #	4 ℃ to 36 ℃
Chemical Stability	Resin is initially stripped at pH ≤ 2.0 followed by buffers with the pH between 6.2 and 8.5. Resin is neutral to salts, sugars and other buffer additives, including protease inhibitors and stays active when caustic buffers are used.
Resin Packing	50% slurry in storage solvent in 18% ± 2% ethanol
Storage	18% ± 2% ethanol, 4 ℃ to 8 ℃

Table 2. Recommended flow rates for 8 x 600 µL i CapTag ^TM high-throughput set of columns (5 mm ID x 30 mm H; V = 600 μL).

Phase	Equilibrate	Bind Wash	ΔpH	Cleaving	Cleaving Incubation	Elution	CIP, Storage [1]
Duration	10 CV [2]	Feed [3]	15 CV	5 CV	3-16 hours [4]	5 CV	10 CV + 10 CV
Flow Rate [microL/min]	600	150 recommended (300 max.)	600	600	0	300	600
Buffers [5]	A1	N/A	A2	B1	N/A	B1	B2:10 CV ST:10 CV
Outlet Fractions	Waste	1 CV	1 CV	1 CV	-	1 CV	B2:1 CV ST: N/A

Legend:

[1] CIP - Cleaning in Place.

[2] CV - Column Volume.

[3] Apply the entire volume of the clarified feed, or the maximum necessary to saturate column.

[4] Dependent on X ⁺¹ and X ⁺² of target protein (See Table 1 in the manual).

[5] Listed in Table 6 in the manual.

Abbreviations & Notes:

* - Specific parameters of the resin are included in the Certificate of Analysis based on the lot number of the resin.

** - The pressure varies based on column tubing and chromatography medium.

& - Note that the first two amino acids of protein of interest have a significant impact on the cleavage rate, where aromatic residues tend to accelerate cleaving while smaller residues slow or stop cleaving and proline abolishes cleaving (see additional instructions). For proteins with unacceptably slow cleaving, amino acids can be added or modified at the N-terminus to provide more optimal cleaving.

# - Note that the temperature effects cleaving. Typically, lower temperatures slow down cleaving rate and higher speed up cleavage.

d _50v - Median particle size of the cumulative volume distribution.

Safety Information

Ethanol, solution: 18% ± 2%

Hazard class: 3

Identification number: UN1170

Packing Groups: PG 3