Capturem™ Protein A

Faster, Easier Antibody and Protein Purification

Capturem Protein A for Antibody Purification and Screening

Capturem His-Tagged Purification for Recombinant Proteins

Combined with the specific antibody-binding properties of Protein A, Capturem technology provides rapid, high-quality purification and screening of monoclonal and polyclonal antibodies, as well as immunoprecipitation and co-immunoprecipitation.

Capturem his-tagged purification columns utilize high-capacity Ni-NTA membranes to quickly purify concentrated recombinant proteins under a wide range of conditions.

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High-throughput screening and process development with 96-well plates Fast, efficient evaluation of conditions for binding, washing, and elution steps 96-well plates for manual or automated systems, with centrifugation or under vacuum Purification and concentration of different isotypes from a variety of source species

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Capturem™ Technology

High-throughput purification of concentrated eluates with 96-well formats Choice of cell lysis system (compatibility with various lysis buffers) Excellent recovery of diluted proteins—concentrate and purify in one step Consistent results regardless of: Native or denaturing conditions Presence of additives (EDTA, bME, TCEP, and DTT)

Monoclonal Cas9 antibody screening from hybridomas

4

5

6

7

8

Clone 1 1

2

Clone 2 1

Clone 3

2

1

2

Clone 4 1

EDTA

2

Clone 5 1

1.

2.

3.

4.

5.

6.

7.

8.

2

Clone 6 1

2

Clone 7 1

Clone 1 = 0.23 mg/ml Clone 2 = 0.27 mg/ml Clone 3 = 0.14 mg/ml Clone 4 = 0.18 mg/ml Clone 5 = 0.30 mg/ml Clone 6 = 0.18 mg/ml Clone 7 = 0.18 mg/ml Polyclonal Ab

2

M

250 – 150 –

1 mM

5 mM

M a Ly rke s r Fl ate ow Fl th ow ro Fl th ug ow ro h W th ug (10 a ro h m W sh ( ug (20 M a 1 h m ) W sh ( 0 m (30 M a 2 M m ) El sh ( 0 m ) M ua 30 M ) El te m ) ua 1 M El te (10 ) u 1 m El ate (20 M ua 1 m ) El te (30 M u 2 m ) El ate (10 M ua 2 m ) te (2 M 2 0m ) (3 0 M m ) M )

3

10 mM

Polyclonal Ab 1

2

Polyclonal

Clone 3

Clone 5 Sample

1 mM EDTA 5 mM EDTA 10 mM EDTA

Screening for the best monoclonal antibody against Cas9 from hybridoma supernatants. Panel A. Crude antibody supernatant was purified with Capturem Protein A, elution was done in a low volume in order to yield a concentrated antibody, and the resulting purified anti-Cas9 antibodies were resolved on an SDS-PAGE gel. Panel B. Cell lysates expressing low and high amounts of Cas9 (Lanes 1 and 2, respectively) were resolved by electrophoresis and transferred to nitrocellulose membranes. These membranes were then blotted using the anti-Cas9 antibodies purified above. Panel C. From these results, two clones were selected for blotting against dilutions of the Cas9 protein.

Amount in Eluate 1

Sample

139 µg 112 µg 98 µg

TCEP

10 mM βME 20 mM βME 30 mM βME

Amount in Eluate 1

171 µg 168 µg 159 µg

M a Ly rke s r Fl ate ow Fl th ow ro Fl th ug ow ro h W th ug (1 a ro h m W sh ( ug (5 M) as 1 h m W h ( mM (10 M) a 5 ) m El sh ( mM M u 1 ) ) El ate 0 m ua 1 M ( ) t 1 El e u 1 m El ate (5 M) ua 1 m El te (10 M) u 2 m El ate (1 M ua 2 m ) te (5 M) 2 m (1 M 0 ) m M )

2

ar Ly ker sa Fl te ow Fl th ow ro Fl th ugh ow ro ( W th ugh 1 m as ro (5 M) u h W ( gh m as 1 m ( M W h ( M 10 ) as 5 m ) m M El h ( M ) ua 10 ) El te mM ua 1 ) ( El te 1 m ua 1 ( M El te 5 m ) ua 1 ( M El te 10 ) ua 2 m ( t El e 1 m M) ua 2 te (5 M) 2 m (1 M 0 ) m M )

1

Additives compatible with Capturem his-tagged protein purification

C

M

M

B

a Ly rke s r Fl ate ow t W hro as ug El h h ua t El e 1 ua t Fl e 2 ow W thr as ou gh El h ua El te 1 ua Fl te 2 ow W th a r El sh oug ua h El te 1 ua te 2

A

DTT

Maximum compatible amount

Reagent MOPS

200 mM

HEPES

200 mM

Tris

200 mM

EDTA

10 mM

b-mercaptoethanol

30 mM

DTT

10 mM

TCEP

5 mM

Guanidine-HCl

6M

Urea

8M

Nonionic detergent (Triton X-100)

2%

Nonionic detergent (Tween 20)

2%

Anionic detergent (SIDS)

1%

Arginine

500 mM

Glycine

100 mM

Histidine

2 mM

Sodium chloride

2M

Imidazole

40 mM

Glycerol

10%

Rapid Protein A column immunoprecipitation Antibody

Lysate Load

Equilibrate 100–400 µl

Bind

Incubate 10 min M ar Ly ker sa Fl te ow W thr as o u El h gh ua te

100–400 µl

Wash

Immunoprecipitation performed with Capturem Protein A columns.

NIH3T3 lysates were incubated with 1 µg of protein phosphatase type 2A (PP2A) B subunit antibody before being applied to equilibrated Capturem Protein A Miniprep spin columns. After washing in 300 µl of wash buffer and eluting in 100 µl of elution buffer, the various fractions were resolved by gel electrophoresis, transferred onto PVDF membranes, and probed with an anti-PP2A antibody. The Capturem Protein A spin columns yielded a strong protein band in the eluate corresponding to the desired PP2A B subunit.

Elute

PP2A B subunit

Analyze

Sample

Amount in Eluate 1

1 mM TCEP

159 µg

5 mM TCEP 10 mM TCEP

209 µg 75 µg

Visit www.clontech.com/hybridoma-screening to view data on rapid screening of hybridoma clones.

Visit www.clontech.com/capturem-IP to view data on fast, efficient IP and Co-IP.

Amount in Eluate 1

1 mM DTT

153 µg

5 mM DTT 10 mM DTT

156 µg 143 µg

Visit www.clontech.com/his-tagged-miniprep to view data on experimental conditions and lysis buffer compatibility.

100 µl

20–50 µl

Sample

Purification of GFPuv in the presence of a wide range of additives, at varying concentrations. Additives were included in the sample, equilibration, wash, and elution buffers for each experiment. Samples were eluted twice with 300 µl of elution buffer each time.

Visit www.clontech.com/secreted-protein to view data on purification of active secreted proteins.

Takara Bio USA, Inc. • A Takara Bio Company United States/Canada: +1.800.662.2566 • Asia Pacific: +1.650.919.7300 • Europe: +33.(0)1.3904.6880 • Japan: +81.(0)77.565.6999 For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. The TaKaRa® logo is a trademark of Takara Holdings Inc. Cellartis® is a trademark of Takara Bio Europe AB. Takara Bio USA, TBUSA, Clontech®, the Clontech logo, Capturem, That’s Good Science!®, and that’s GOOD science! stylized form® are trademarks of Takara Bio USA, Inc. All other trademarks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions. ©2016 Takara Bio USA, Inc.

09.16 US (633830A)

www.clontech.com

High-capacity membranes: no resin, no incubation

Capturem Technology Makes Resin Obsolete

Fast protocol (5–15 min) No resin, no incubation

High-Quality Results Without the Effort

The Capturem Family: Fast, Efficient Purification

Capturem purification kits are a revolutionary solution for recombinant protein and antibody purification. With a unique combination of established chemistry and gamechanging technology, the Capturem family of products effortlessly outperforms conventional methods:

Capturem kits use disposable affinity columns or plates containing novel high‑capacity Ni2+ or Protein A nylon membranes for quick and easy purification of his‑tagged proteins or antibodies, respectively.

Get high-quality, concentrated proteins and antibodies without the time and effort of resin-based methods. Capturem technology brings speed and flexibility to your protein purification workflow.

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Rapid, resin-free protocols performed at room temperature Concentrated eluates for direct use in downstream applications Purification from mammalian or bacterial cells Easy handling of proteins that are otherwise difficult to purify Compatibility with additives and media (no buffer exchange needed)

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High-capacity membranes Traditional membrane pores suffer from a lack of available surface area, and resin column protocols require several hours to complete. However, our proprietary modified membranes have a Resin columns much higher surface area—and therefore a higher protein binding capacity—yet provide high-quality results in just minutes.

Concentrated, high-quality eluates Compatible with additives & media

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Visit our website to explore our ever-growing portfolio of Capturem products. Browse learning resources and technical information:

• New products • Visual protocols • Technical notes • Videos

Capturem purification protocol

- High capacity (surface area) - Slow diffusion of macromolecules within pores - Long separation times

Capturem membrane pores

Fast and convenient: Minipreps are completed in five minutes, and maxipreps and 96‑well plate purifications are completed in less than 15 minutes, without additional incubation.

High-throughput format: 96-well plates are appropriate for manual or automated systems, and compatible with centrifugation or vacuum.

Concentrated eluates: A small bed volume enables elution at high concentrations.

Purity: The small bed volume traps fewer contaminants, and wash buffer at 300X the bed volume allows for more thorough washing.

Flexibility: Columns are compatible with additives and mammalian media; no buffer exchange is needed before loading samples.

Downstream success: Short residence times reduce the possibility of antibody aggregation, protein degradation, and loss of activity.

His-tagged protein purification

Resin columns

High capacity (large internal • High capacity (large internal surface area) Traditional membrane pores surface area) Small bed volume • Large bed volume Low capacity internal surface area)of macromolecules Rapid flow-induced mass transport • (small Slow diffusion - Rapid flow-induced mass transport pores - Low pressure within drop Short residence time • Long residence times

Antibody purification

Lyse cells

Antibody Sample

2−5 ml culture Up to 800 µl lysate

Up to 800 µl

Capturem His-Tagged Purification Miniprep Kit and Capturem Protein A Miniprep workflows. Each mini spin column can be loaded with up to 800 µl of lysate (yielded from 2–5 ml of culture) or diluted antibody sample (diluted from 1:1 to 1:20 with buffer). Each step is followed by a one-minute spin, and the entire miniprep purification is completed in five minutes.

The working bed volume of the membrane is <2 µl, and over 90% of the bound protein or 80% of the bound antibody can be eluted with as little as 100 µl of elution buffer.

Load

Equilibrate Resin columns

Modified membrane pores Resin columns

- High capacity (large internal surface area) - High capacity (surfacemass area) - Rapid flow-induced transport - Low pressure - Slow diffusion ofdrop macromolecules within pores

400 µl

Bind

- High capacity (surface area) - Slow diffusion of macromolecules within pores - Long separation times

up to 800 µl

Wash

- Long separation times

only 5 min

Learn more:

300 µl

Capturem Ni membrane for his-tagged proteins 2+

Traditional resin columns Traditional membrane pores

www.clontech.com/capturem

Elute

Traditional membrane pores

100−300 µl - High capacity (large internal surface area) - Rapid flow-induced mass transport - Low pressure drop

- Low capacity (small internal surface area) - Rapid flow-induced mass transport - Low pressure drop

Analyze h gh ug u 1 1 2 2 r e ro ro ke pl wth wth sh sh tion tion tion tion r a m o a a M Sa Flo Fl W W Elu Elu Elu Elu

Modified membrane pores

M 1 2 3

Capturem Protein A membrane for antibodies

4 5

h gh ug u r e ro ro ke pl wth wth sh sh tion tion r a m o a a M Sa Flo Fl W W Elu Elu

6 7 8 9

- High capacity (large internal surface area) - Rapid flow-induced mass transport - Low pressure drop

Visit www.clontech.com/capturem for more information

Visit www.clontech.com/capturem for more information

www.clontech.com/capturem