Anti-Myc-Tag Mouse mAb (Agarose Conjugated) is a monoclonal anti-Myc antibody covalently linked to agarose; the agarose enables immunoprecipitation (IP) of Myc tagged proteins or co-immunoprecipitation (Co-IP) of their interacting partners.

This Abmart monoclonal antibody is produced by immunizing animals with a synthetic peptide (KLH-coupled) corresponding to esidues 410-419 of human c-Myc (EQKLISEEDK).

Mouse IgG1

Anti-Myc-Tag Mouse mAb detects transfected proteins containing the Myc epitope tag.

All

0.2 M Glycine, pH 2.5

The work can be performed in 1.5 ml micro-centrifuge tubes or in spin columns.

1. Thoroughly resuspend the Anti-Myc Agarose by inverting the tube or vial several times.

2. Add 20-50μl 50% slurry of Anti-Myc Agarose into cell lysate using a widebore pipette tip.

Note: The lysate should be fresh, and for a well expressed tagged protein, 200μl lysate (200-500μg total protein) usually yields a good IP result.

3. Incubate with gentle mixing for 2 h to overnight at 4°C.

4. Wash the beads with 1 ml TBS buffer or lysis buffer, such as RIPA (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium

deoxycholate), centrifuge for 3 min at 2,000× g, and discard the supernatant.

Wash 3 times, avoid losing beads during washes.

5. Elution of the Myc tagged protein.

Option 1. Elution with elution buffer.

Add 30-50 l elution buffer to the beads, gently tap the tube to mix well, centrifuge for 3 min, transfer the supernatant very carefully to

a fresh tube (Avoid transferring any beads).

Note: Neutralize the eluant immediately by add 1 l of 1.5 M Tris, pH 9.0 per 20μl Elution buffer.

Option 2. Elution with Myc peptide

Add 30-50μl Myc peptide solution (100μg/ml HA peptide in TBS buffer), gently tap the tube to mix well, incubate for 10 min, centrifuge for 3 min,

and transfer the supernant to a fresh tube. TBS buffer: 50 mM Tris HCl, 150 mM NaCl, pH 7.4.

Option 3. Elution with SDS loading buffer

Add 30μl 2×SDS loading buffer, gently tap the tube to mix well, boil at 100 for 5 min, centrifuge for 3 min, transfer the supernatant to a fresh

tube.

Note: in this case, the supernatant contains not only the binding proteins, but also IgG (heavy and light chains).

6. Prepare SDS-PAGE gel for western blotting or proceed to other assays.

The product is supplied as a 50% slurry in storage buffer (1 PBS, pH 7.4, containing 0.1% NaN ). Store the product at 4°C and do not freeze.

AB_2936241

#M20001 His-Tag (2A8) Mouse mAb

#M20002 Myc-Tag (19C2) Mouse mAb

#M20003 HA-Tag (26D11) Mouse mAb

#M20004 GFP-Tag (7G9) Mouse mAb

#M20007 GST-Tag (12G8) Mouse mAb

#M20008 DYDDDDDK-Tag (3B9) Mouse mAb (Binds to same epitope as

Sigma’s Anti-FLAG M2 Antibody)

#M20013 Anti-HA-Tag Mouse mAb (Agarose Conjugated)

#M20018 Anti-DYKDDDDK-Tag Mouse mAb (Agarose Conjugated) (Binds to

same epitope as Sigma’s Anti-FLAG M2 Antibody)