Myc-Trap for both immunoprecipitation and affinity purification of Myc-tagged proteins

The Alpaca antibody advantage for pull-down of Myc-tagged proteins

Practical considerations and instructions

Introduction

The ChromoTek  Myc-Trap®  is an affinity resin that consists of Myc-binding  protein derived from an Alpaca single domain antibody, also called  anti-Myc VHH or “anti-Myc nanobody”, which is coupled to agarose and magnetic agarose beads. The Myc-Trap provides advantages over conventional IgG antibodies when applied in immunoprecipitation (IP)  and affinity purification experiments:

1. no contamination of the protein of interest by heavy or light antibody chains (which may be the case when conventional antibodies are used as affinity resin)
2. high specificity, sensitivity and affinity binding of the protein of interest enabling the pulldown or purification of low expressed/abundant proteins at high reproducibility
3. gentle and effective elution to obtain native protein of interest
    

Optimize performance of your experiment

Myc-Trap’s performance has been optimized for immunoprecipitation (IP/Co-IP) and affinity purification of Myc-tagged proteins, which depends on whether you use a single Myc-tag, i.e. 1xMyc = EQKLISEEDL or a double Myc-tag fused to the protein of interest, i.e. 2xMyc = EQKLISEEDLEQKLISEEDL. Although both IP/Co-IP and protein purification can be done with all Myc-constructs, you can optimize your experimental performance by using either either a 1xMyc tagged or a 2xMyc-tagged protein. For an overview see table 1:

1xMyc-tagged protein for effective & gentle elution	2xMyc-tagged protein for effective binding
Gentle elution of native fusion protein One step elution Small volume to concentrate fusion protein	Low expressed/concentration proteins Complete binding Extraordinary high binding affinity Stringent wash conditions for high pure results

Table 1: Summary  Myc-Trap as affinity resin for immunoprecipitation and affinity purification: Binding of 1xMyc and 2xMyc-fusion proteins results in application optimized performances. Note: Both IP/Co-IP and affinity purification can be done with all Myc constructs.

Description of binding and elution optimized performances

ChromoTek has investigated the binding mechanism of Myc-Trap in order to validate the function of this affinity resin. As a result, 2xMyc-tagged proteins are tighter bound to Myc-Trap than Myc-fusion proteins, which is manifested in different dissociation constants K_D. These result from different binding mechanisms (for an introduction and discussion of the Myc-Trap’s epitope, see whitepaper  How to plan an IP of your Myc-tagged protein when using the ChromoTek Myc-Trap).

Kinetic paramters are shown for 1xMyc-Maltose Binding Protein (MBP) and 2xMyc-MBP in table 2. Myc-Trap binds 1xMyc-tagged MBP tightly with a low K_D  of 400nM. This K_D  value already matches general requirements of immunoprecipitation. However, 2xMyc-tagged MBP is about 1,000 times stronger bound with a K_D  of just 0.5nM. These kinetic values explain the high pulldown and binding effectiveness of 2xMyc-tagged protein and the gentle elution capability of Myc-tagged protein when using Myc-Trap.

Because of the fast binding of 1xMyc- and 2xMyc-fusion proteins to the affinity resin Myc-Trap (high binding rates k_on), the binding of Myc-fusions is already completed within 30 minutes. However, the low dissociation rate (k_off) of bound 2xMyc-fusion proteins allows for prolonged incubation and washing, whereas the relative high dissociation rate (k_off) of Myc-fusion protein requires instant washing with short durations.

Kinetic parameter	1xMyc-MBP	2xMyc-MBP
Dissociation constant KD	400nM	0.5nM
Binding rates kon	5.3 x 10^5 M^-1 s^-1	2.9 x 10^5 M^-1 s^-1
Dissociation rate koff	2.1 x 10^-1 s^-1	1.4 x 10^-4 s^-1

Table 2: Kinetic parameters of 1xMyc and 2xMyc-fusion proteins bound to Myc-Trap  Experiments were conducted using Maltose Binding Protein (MBP) fused to Myc-tag. The K_D  value for binding 1xMyc-MBP indicates strong binding and is in range what is generally required for IP. It does allow gentle and effective elution. The K_D  value for 2xMyc-MBP is about 1,000 fold smaller, indicating a considerable increase of affinity and binding strength and demonstrating the outstanding IP performance of Myc-Trap. It does enable binding of 2xMyc-tagged proteins at low concentrations and under harsh binding conditions.

There are various elution options dependent on your experimental strategy, i.e. whether you require effective/gentle elution or effective pull-down/binding. Four elution options and their performances are presented in table 3. As you can see, the effectiveness of these options actually depends on whether you immunoprecipitate/purify 1xMyc- or 2xMyc-tagged protein.

• If you like to effectively or gently elute your protein of interest in native conditions use 1xMyc-tagged protein in your experiement
• If you like to effectively pull-down/bind low expressed/abundant proteins, or low concentration proteins use 2xMyc-tagged protein in your experiment

Elution with	Elution Effectiveness
	1xMyc-MBP	2xMyc-MBP
2xMyc-peptide (40 µM)	+++	+
Urea (8 M)	+++	+
Glycine (0.2 M) pH 2.5	+	+

Table 3: Effectiveness of Myc-Trap elution options.  Experiments were conducted using 1x and 2xMyc-tagged Maltose Binding Protein (MBP). Instead of 2x Myc-tag also 3xMyc-tagged protein can be used. Abbreviations:
o: <50%, +: ca. 50%, ++: ca.70%, +++ ca. 90% of bound protein eluted.

Afore data shall enable you to consider the optimal conditions for your experiment with Myc-Trap. Nevertheless, a summary of experimental design considerations is presented in table 4. If you like to obtain more detailed information on discussed matter or information about the Myc-Trap’s epitope and how Myc-Trap binds 1xMyc- and 2xMyc-fusion proteins please refer to our whitepaper How to plan an IP of your Myc-tagged protein when using the ChromoTek Myc-Trap.

1xMyc-tagged protein	2xMyc-tagged protein
Effective one step elution: both 2xMyc-peptide and urea result in fairly complete elution of bound Myc-tagged protein 2xMyc-peptides enable gentle elution to obtain native Myc-tagged proteins Small elution volume to concentrate Myc-fusion protein of interest Fast binding for short incubation time Fast dissociation requires quick wash processes	Stringent wash condition can be applied Effective binding of low or endogenous expressed 2xMyc-fusion protein Complete pulldown/binding Incomplete elution favors on-bead downstream assays & digestion of 2xMyc fusion protein Fast binding enables short incubation of 2xMyc fusion protein Slow dissociation of 2xMyc-fusion protein enables long wash steps

Table 4: Summary of experimental design considerations

For more information