Immunoprecipitation-Protein-A-G

Magnetic Separation Technology

It’s time to update your immunoprecipitation

Dynabeads? Protein A and Protein G

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Magnetic Separation Technology

The new gold standard for immunoprecipitation Dynabeads? Protein A and Protein G

Reduce protocol time to 30 minutes

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Isolate intact proteins and protein complexes

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Eliminate background caused by nonspecific binding

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New kits with premade buffers for convenience and increased consistency

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You need only a Dynabeads? Protein A or Protein G Immunopre-

cipitation Kit, your specific antibody of choice, and a DynaMag?

magnet (Figure 1). The protocol takes place in a single tube with

just a few handling steps (Figure 2). The attraction is simply

magnetisk.*

Fast and gentle

Immunoprecipitation with Dynabeads? is fast and gentle, caus-

ing minimal physical stress to target proteins. Rapid kinetics

and short incubation times reduce the protocol time to only

30 minutes.

Your target proteins no longer need to enter the interior of

porous resins. Gentle magnetic handling permits the isolation of

labile complexes that might otherwise dissociate or be damaged

by proteases during long incubations. Native protein conforma-

tion and intact, large protein complexes are preserved.

Easy, flexible, and automatable

The protocol can be scaled to match your specific purpose and

sample volume requirements. By scaling down, you can easily

reduce the consumption of expensive antibodies.

* M agnetisk is the Norwegian word for magnetic. Did you know that magnetic separation technology was pioneered in the 1980s by the Norwegian company Dynal, now part of Life Technologies? To learn more, check out https://www.sodocs.net/doc/903437438.html,/dynal.

An important advantage of short incubation times and the

low nonspecific binding characteristics of Dynabeads? is that

there is no need for time-consuming preclearing or dilution. Say

good-bye to liquid chromatography, columns, filters, centrifuges,

or other complicated equipment. The consistent size and shape

of the Dynabeads? (Figure 3) ensure consistent and predictable

behavior, and the protocol is easily automated on a liquid han-

dling platform.

Efficient and reliable

Gentle magnetic handling allows you to work with concentrated

protein solutions throughout the procedure. You do not need to

worry about losing material from spun-down resins or excess sur-

face during pipetting. Washing and elution are very efficient and

ensure maximal sensitivity, minimal loss of target protein, and no

background caused by nonspecific binding.

Dynabeads? are monodisperse (Figure 3) and feature optimal

accessibility and binding kinetics (Figure 4). Convenience and con-

sistency are further strengthened by the ready-made buffers in the

kits. Immunoprecipitation with Dynabeads? allows you to reduce

variability and provides absolute reproducibility for your research.

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A B

Figure 1—New magnets enable efficient immunoprecipitation. The new DynaMag?-2 (A) and DynaMag?-Spin (B) magnets combine strong magnetic attraction with flexible and efficient ergonomic design. Sample racks from both magnets can be removed from the magnet base for vortexing or manual handling of your sample vials.

Figure 2—Immunoprecipitation in only 30 minutes. Dynabeads? precoupled with protein A or protein G act as a suspendable solid support that can be fixed by the use of a magnet. This allows for simple and efficient antibody capture, fol-lowed by immunoprecipitation of your pure target peptides, proteins, protein complexes, or other antigens.

Y Y

Y

Y Y

Y

Y

Y Y

Mild elution for isolation of native protein

Denaturing elution for isolation of denatured target protein

Figure 3—Dynabeads? reduce variability in your research. (A) Uniform, monodis-perse superparamagnetic Dynabeads? are manufactured with highly control-lable product qualities and to a unique level of reproducibility within and between batches. (B–D) Magnetic particles from alternative suppliers.

Table 1—Binding characteristics of different immunoglobulins (Ig). Native protein G and protein A differ in their binding to Ig classes from different species and sub-classes. For example, human IgG3 will bind strongly to protein G, but only weakly to Protein A. S: strong binding; M: medium binding; W: weak binding; N: no binding.

Species

Ig class Protein A

Protein G

Human

Total Ig S S IgG1, IgG2, IgG4

S S IgG3W S IgD N N IgA, IgM W N Fab W W ScF v W N Mouse

Total Ig S S IgG1

W M IgG2a, IgG2b, IgG3S S IgM N N Rat

Total Ig W M IgG1W M IgG2a N S IgG2b N W IgG2c S S Goat

Total Ig W S IgG1W S IgG2S S Sheep

Total Ig W S IgG1W S IgG2S S Cow

Total Ig W S IgG1W S IgG2S S Horse

Total Ig W S IgG(ab)W N IgG(c)W N IgG(T)N S Rabbit Total Ig S S Dog Total Ig S W Cat Total Ig S W Pig

Total Ig S W Guinea pig Total Ig S W Chicken

Total Ig

N

N

DYNAL? has pioneered magnetic separation technologies for biological discovery that are both simple and highly reproducible. Based on their patented superparamagnetic, monodisperse beads, Dynabeads? technologies represent a superior paradigm for cell and biomolecule separation in a wide range of basic and clinical research applications, diagnostic assays, and therapeutic protocols.

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?2009 Life Technologies Corporation. All rights reserved. Trademarks of Life Technologies Corporation and its affiliated companies: Dynabeads ?, Dynal ?, DynaMag?, Invitrogen?. All other trademarks are the sole property of their respective owners. These products may be covered by one or more Limited Use Label Licenses (see Invitrogen catalog or https://www.sodocs.net/doc/903437438.html,). By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated. B-081910 0109

Which product suits your research best?

Protein A and protein G have high specificity for immunoglobu-lins (Igs). The specific binding strength will depend on the species and Ig subclass (Table 1). Dynabeads? Protein A and Dynabeads?

Protein G have a binding capacity of 250 μg human IgG per millili-ter of beads. Predominant Fc binding allows optimal Ig orientation and enhances performance in immunoprecipitation.

Ordering information

Product

Quantity

Cat. no.

Immunoprecipitation Kit —Dynabeads ? Protein A

Complete kit with Dynabeads ? Protein A (2 ml) and required buffers 1 kit (40 IP reactions)100-06D Dynabeads? Protein A

1 ml (~40 mg/ml)100-01D Magnetic beads coupled with recombinant protein A

5 ml (~40 mg/ml)100-02D Immunoprecipitation Kit —Dynabeads ? Protein G

Complete kit with Dynabeads ? Protein G (2 ml) and required buffers 1 kit (40 IP reactions)100-07D Dynabeads? Protein G

1 ml (~30 mg/ml)100-03D Magnetic beads coupled with recombinant protein G 5 ml (~30 mg/ml)100-04D DynaMag?-2

1 unit

123-21D

Magnet holding up to 16 standard 1.5–2 ml microcentrifuge tubes. Working volume: 10–2,000 μl DynaMag?-Spin

1 unit

123-20D

Magnet holding up to 6 standard 1.5 ml microcentrifuge tubes. Working volume: 10–1,500 μl

Figure 4—Shorter protocol time and better yields with Dynabeads?. The same input of antibodies (Ab) and cell lysate was used for all IP protocols. With Dynabeads? Protein A (A) and Dynabeads? Protein G (B), all the antibodies on the bead surface are accessible for optimal, highly reproducible antigen binding.

Antigen (HSA) Ab heavy chain

Ab light chain

Protocol times:

104 min 127 min 63 min

44 min

34 min

60 M W

L y s a t e S e p h a r o s e ? s p i n c o l u m n

S e p h a r o s e ? s l u r r y

A g a r o s e s l u r r y & s p i n c o l u m n

R e s i n s p i n c o l u m n

D y n a b e a d s ? P r o t e i n A

50 40 30 20

Antigen (HSA) Ab heavy chain

Ab light chain

Protocol times:

104 min 127 min 63 min

44 min

34 min

60 M W

L y s a t e

S e p h a r o s e ? s p i n c o l u m n

S e p h a r o s e ? s l u r r y

A g a r o s e s l u r r y & s p i n c o l u m n

R e s i n s p i n c o l u m n

D y n a b e a d s ? P r o t e i n G

50 40 30

A

B

A selection of surface-activated Dynabeads?, and Dynabeads? with streptavidin or secondary antibodies coupled to their sur-face, are also available. To see our full range of Dynabeads? for immunoprecipitation, and to explore further details, protocols,

and references, please visit https://www.sodocs.net/doc/903437438.html,/immunopre-cipitation . See https://www.sodocs.net/doc/903437438.html,/antibodies to find antibod-ies validated for immunoprecipitation.