CaptureSelect™ C-tag Affinity MatrixCatalog Number 191307005, 191307010, and 191307050Pub. No. MAN0010706 Rev. A.0

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Product descriptionThe CaptureSelect™ C-tag Affinity Matrix combines unique selectivityfor a small 4-amino-acid “C-tag” peptide tag E‑P‑E‑A (glutamic acid -proline - glutamic acid - alanine) with the benefits of a robust andhigh-quality affinity matrix provided by a 13 kDa Camelid antibodyfragment.The CaptureSelect™ C-tag Affinity Matrix recognizes the E-P-E-Apeptide when this tag is fused directly to the C-terminus of a protein(see Figure 1).

Figure 1 Representation of a CaptureSelect™ C-tag peptide (E‑P‑E‑A),genetically fused at the C terminus of a recombinant protein.

Note: The Alanine residue (A) of the E-P-E-A sequence needs toremain “free” in order to facilitate proper binding of theCaptureSelect™ C-tag Affinity Matrix. You can incorporate linkersbetween the protein and the C-tag as long as the E-P-E-A sequence isdisplayed at the C-terminal end of the protein of interest.

Product advantagesEpitope tagging is a well-known technology used to facilitate thepurification and detection of recombinant proteins for which nosuitable ligand is available. The technology allows high-selectivecapture, but has limitations, especially when you use large tags thatcan alter protein function, such as GST and MBP. For this reason, thetechnology is usually used with smaller sequences such as hexahistidine- or FLAG-tag. The hexa histidine-tag, however, often lacksgood purity when the protein of interest is extracted from complexmixtures using immobilized metal ion chromatography (IMAC).Peptide tag-antibody systems, such as FLAG-tag, provide a higherselectivity, but are hampered in reusability, due to the poor stability ofmonoclonal antibodies in a chromatographic set-up, and inefficientbinding of target proteins under denaturing conditions such as 8 Murea.The CaptureSelect™ C-tag Affinity Matrix purifies C-terminal taggedproteins with high affinity and selectivity, even in the presence of urea

and guanidine HCl, from complex mixtures like cyto - or periplasmicfractions in a one step process. Mild elution conditions at neutral pHcan be applied using magnesium chloride or propylene glycol, whichensures high activity recoveries of pH-sensitive target proteins.The unique features of the CaptureSelect™ C-tag system make thesystem an excellent alternative to sequences such as His - and FLAG-tag, especially for use in a generic purification strategy within a high-throughput protein production environment. The CaptureSelect™ C-tag Affinity Matrix offers:• High recovery and purity in a single step• Binding under denaturing conditions• Mild elution to retain the biological activity of C-tag fusion

proteins• Compatibility with FPLC systems

Specifications

Ligand CaptureSelect™ C-tag

Binding specificity C-terminal E-P-E-A

Matrix and particle size Aldehyde activated agarose, 35 µm

Dynamic binding capacity ±250 nmol/mL

Shipping solution 20% (v/v) ethanol

Conditions for use

Parameter Conditions for use

Equilibration/wash buffer • Physiological — 20 mM Tris or PBS, pH7.0 to 7.4

• Denaturing —– 50 mM Tris, up to 8 M Urea, pH 7.0–

7.4– 50 mM Tris, up to 1 M guanidine, pH

7.0–7.4

Elution buffer • Neutral (pH 7.0–7.4):– 20 mM Tris, 2.0 M MgCl2– 20 mM Tris, 1 M NaCl, 50% (v/v)

propylene glycol (PG)– 20 mM Tris, 2 mM “S‑E‑P‑E‑A”

peptide, ±NaCl or MgCl2• Acidic:

– 20 mM citric acid, pH 3– 100 mM glycine, pH 3–2

Flow rate 100–200 cm/hr

Pressure limit £2 bar

For Research Use Only. Not for use in diagnostic procedures.

Parameter Conditions for use

Cleaning solution Any of the following, prepared freshlyevery 2–3 days and stored protected fromlight to minimize radicals that affect thefunctionality of the matrix:• Citric acid or acetic acid, 0.5–1 M• 10 mM NaOH, pH 12• PAB (120 mM phosphoric acid, 167 mM

acetic acid, and 2.2% (v/v) benzylalcohol)

Storage solution andtemperature

20% (v/v) ethanol• Short term: Room temperature• Long term: 4°C for up to 2 years

Guidelines for use - Spin columnsThe following method is for small-scale purification or analyticalpurposes using disposable spin columns and common laboratoryequipment (see Figure 2).

Figure 2 Scheme for protein isolation using a spin-column format.

For optimal matrix performance, optimize the conditions in theguidelines below for your application.1. Prepare a 1:1 slurry of matrix and storage solution (20% v/v

ethanol). Carefully mix (invert the matrix container or use apaddle stirrer) to create a homogeneous distribution.

Note: We do not recommend the use of mechanical stirrers,homogenizers, or similar devices.

2. For 100 µL of matrix, carefully apply 200 µL of the CaptureSelect™

Affinity Matrix slurry (from step 1) to the spin column (useMoBiTec Mobicol F spin columns or the equivalent), then spin for1 minute at ~700 × g (~3,000 rpm in an Eppendorf centrifuge).

3. Add 500 µL equilibration buffer to wash the matrix, then spin for1 minute at ~700 × g. Repeat this step two times (three washes intotal).

4. Load 100–700 µL of the protein C-tag-containing sample on theaffinity matrix in the spin column. Close the spin column andincubate for 30–60 minutes on a rotating wheel.

5. Collect the non-bound proteins (flow-through) in a 2-mL collectiontube by centrifuging the spin column for 1 minute at ~700 × g.

6. Wash the matrix 2–4 times as described in step 3.7. Elute the captured C-tag fusion protein by adding 200–400 µL

elution buffer, then centrifuge at 700 × g to collect the elutionfraction.

Note: For optimal recovery, you may need to repeat the elutionstep one or more times.

8. (Optional) Strip the CaptureSelect™ Affinity Matrix [for example,with 100 mM glycine (pH 2) followed by re-equilibration inequilibration/wash buffer] to allow a second spin columnpurification run.

9. If you will not use the spin column immediately, store the matrixin 20% ethanol at 4°C.

Note: For more information about MoBiTec spin columns please visit www.mobitec.de

Example application using spin columnsThe CaptureSelect™ C-tag Affinity Matrix provides excellent selectivityfor purification of recombinant proteins equipped with the C-tag fromcomplex mixtures, such as cyto- or periplasmic fractions from E.coli-derived expression systems.In Figure 3 and Figure 4, crude samples were applied in disposablespin columns, each containing 100 µL of the CaptureSelect™ C-tagAffinity Matrix. After incubating and washing the samples, the boundC-tag fusion proteins were eluted using a neutral pH-based elutionbuffer (20 mM Tris, 2 M MgCl2, pH 7.0), followed by a column strip atpH 2. The results demonstrate very good target recovery and purityfrom complex mixtures in a one-step process using a neutral pH-basedelution buffer.

Figure 3 Purification of a Camelid domain antibody equipped witheither the C-terminal tag -GYQDY-E‑P‑E‑A (A) or -GAA-E‑P‑E‑A (B), in aspin column format from E.coli-derived periplasmic fractions (400 µLperi/100 µL affinity matrix per spin column). KEY: ST: startingmaterial, FT: flow-through, Elution: 20 mM Tris, 2 M MgCl2, pH 7.0

Figure 4 Purification of a green fluorescent protein (GFP) equippedwith the C-terminal tag -GYQDY-E‑P‑E‑A in a spin column format froman E.coli cytoplasmic fraction (400 µL crude sample/100 µL affinitymatrix per spin column. KEY: ST: starting material, FT: flow-through,W: wash, Elution: 20 mM Tris, 2 M MgCl2, pH 7.0

Note: For very sensitive proteins that are not compatible with either ofthe suggested mild elution buffers, you can release the capturedprotein by competition (for example, by using the synthetic peptide"S‑E‑P‑E‑A" at a concentration of 2 mM in 20 mM Tris, pH 7). Morethan 60% release is observed using these conditions, which you canfurther optimize by adding NaCl and/or increasing the S-E-P-E-Apeptide concentration.

2 CaptureSelect™ C-tag Affinity Matrix Product Information Sheet

About binding under denaturing conditionsThe CaptureSelect™ C-tag system provides a high level of selectivity,and also allows the protein of interest to be bound under denaturingconditions. After the protein is bound to the matrix (for example, inthe presence of 8 M urea), you can incubate the affinity matrix-suitablebuffer to allow on-column re-folding before elution.

Figure 5 Elution fractions of a C-taged protein, after binding to the C-tag affinity matrix using different concentrations of denaturing agents(urea or guanidine-HCl). After re-folding in PBS, the captured proteinwas eluted using 100 mM glycine pH 3.

Guidelines for use - FPLCFor optimal matrix performance, optimize the conditions in theguidelines below for your application.1. Carefully pack the CaptureSelect™ C-tag Affinity Matrix in a

column, then equilibrate the matrix by adding equilibration/washbuffer.

2. Attach the packed column to the FPLC system.3. Equilibrate with 10 CVs of equilibration/wash buffer.4. Determine the volume of sample to load, based on the dynamic

binding capacity (±250 nmol/mL), concentration of the targetmolecule, and the column size. Optimum loading is atphysiological pH. Avoid acidic conditions which decrease bindingefficiency.

5. Load the sample on the column.6. Wash the sample with 5–10 CVs of equilibration/wash buffer. You

can increase the NaCl or MgCl2 concentration up to 1.0 M tooptimize washing efficiency.

7. Elute with 3–5 CVs of elution buffer.8. Re-equilibrate the column in equilibration/wash buffer.9. Strip the column with 0.1 M glycine (pH 2.0), citric acid, or acetic

acid (0.5–1.0 M).10. Re-equilibrate the column in equilibration/wash buffer to prepare

the column for another purification run.11. If the column will not be used immediately, the matrix should be

stored in 20% ethanol at 4°C.

Column cleaning guidelines1. Pump cleaning solution through the column for 15 minutes.2. Pump 5–10 CVs equilibration/wash buffer to re-equilibrate the

column.

Example application using FPLCYou can use the CaptureSelect™ C-tag Affinity Matrix in a packed-bedformat to provide good binding capacities at high flow rates (that is,150 cm/hr). The examples (Figure 6 and Figure 7) illustrate thepurification of a Camelid domain antibody equipped with a C-terminal E‑P‑E‑A tag.

Figure 6 Purification of a Camelid domain antibody equipped with aC-terminal His6-E‑P‑E‑A tag from E.coli periplasmic fraction,demonstrating that CaptureSelect™ C-tag Affinity Matrix outperformstwo different Ni-NTA based resins on protein purity in a one-steppurification process (analyzed by SDS-page).

Figure 7 Purification of a Camelid domain antibody equipped with aC-terminal GAA-E‑P‑E‑A tag on a 400 µL Tricorn column (2‑cm bedheight volume) using an Äkta Explorer system.

Red line: OD280 signal; Grey line: pH value

Protein C-tag detection and quantitationIn addition to the CaptureSelect™ C-tag Affinity Matrix, the anti-C-tagaffinity ligand is also available as a Biotin ligand conjugate(CaptureSelect™ Biotin Anti-C-tag Conjugate) in order to facilitate theeasy detection and/or quantitation of C-tag fusion proteins in, forexample, ELISA, Western blot, and label-free platforms such as thosebased on surface plasmon resonance (Biacore® and IBIS-MX96systems) and bio-layer interferometry (ForteBio® Octet® systems). Seethe CaptureSelect™ Biotin Anti-C-tag Conjugate Product Information Sheet(Pub. no. MAN0010067).

Ordering Information

Product Cat. no.

CaptureSelect™ Biotin Anti-C-tagConjugate

7103252100 (100 μg)

7103252500 (500 μg)

CaptureSelect™ C-tag Affinity Matrix 191307005 (5 mL)

191307010 (10 mL)

191307050 (50 mL)

For more informationFor more information on CaptureSelect™ products and ligand leakageELISA products, go to www.lifetechnologies.com/captureselect.

CaptureSelect™ C-tag Affinity Matrix Product Information Sheet 3

Obtaining SDSsSafety Data Sheets (SDSs) are available from www.lifetechnologies.com/support.

Note: For the SDSs of chemicals not distributed by Thermo FisherScientific, contact the chemical manufacturer.

Limited product warrantyLife Technologies Corporation and/or its affiliate(s) warrant theirproducts as set forth in the Life Technologies' General Terms andConditions of Sale found on Life Technologies' website at www.lifetechnologies.com/termsandconditions. If you have anyquestions, please contact Life Technologies at www.lifetechnologies.com/support.

ReferencesDjender, S. et al. 2014. The Biotechnological Applications ofRecombinant Single-Domain Antibodies are Optimized by the C-Terminal Fusion to the EPEA Sequence (C Tag). Antibodies 3:182–191.

De Genst, E.J. et al. 2010. Structure and properties of a complex ofα-synuclein and a single-domain camelid antibody. J Mol Biol.402(2):326–43.

For Research Use Only. Not for use in diagnostic procedures.

The information in this guide is subject to change without notice.

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25 June 2014