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

This Abmart monoclonal antibody is produced by immunizing animals with a synthetic peptide containing epitope DYKDDDDK (KLH-coupled).

Mouse IgG2b

Anti-DYKDDDDK-Tag Mouse mAb detects transfected proteins containing the DYKDDDDK 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-DYKDDDDK Agarose by inverting the tube or vial several times.
2. Add 20-50ul 50% slurry of Anti-DYKDDDDK Agarose into cell lysate using a wide-bore pipette tip.
Note: The lysate should be fresh, and for a well expressed tagged protein,
200ul lysate (200-500ug 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 DYKDDDDK tagged protein.
Option 1. Elution with elution buffer.
Add 30-50 μl elution buffer to the beads, gently tap the tube to mix well, immediately centrifuge for 3 min, transfer the supernatant very carefully to a fresh tube (Avoid transferring any beads).
Note: Neutralize the eluant immediately by add 1μl  of 1.5 M Tris, pH 9.0 per 20 μl Elution buffer.
Option 2. Elution with DYKDDDDK peptide
Add 30-50 μl DYKDDDDK peptide solution (100 μg/ml DYKDDDDK 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 30 μ l X  2 SDS loading buffer, gently tap the tube to mix well, boil at 100°C 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_2936247

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