DYKDDDDK Epitope Tag Mouse Monoclonal Antibody [Clone ID: 3B9]

CAT#: AM20780AG-N

DYKDDDDK Epitope Tag mouse monoclonal antibody, clone 3B9, Agarose

Specifications

Product Data

• Clone Name: 3B9
• Applications: IP
• Recommended Dilution: Immunoprecipitation.
• Reactivities: All Species
• Host: Mouse
• Isotype: IgG2b
• Clonality: Monoclonal
• Immunogen: Synthetic peptide containing epitope DYKDDDDK (KLH-coupled).
• Specificity: This antibody reacts to DYKDDDDK Epitope Tag.
• Formulation: 50% slurry in storage buffer (1× PBS, pH 7.4, containing 0.09% sodium azide).
  Recommended elution buffer: 0.2 M Glycine, pH 2.5
  Label: Agarose
  State: Liquid purified Ig fraction
• Conjugation: Agarose
• Background: 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 co-immunoprecipitation (Co-IP) of their interacting partners.
• Synonyms: FLAG Epitope Tag, ECS Epitope Tag, FLAG-tag, ECS-tag, D-tag
• Note: Protocol: Immunoprecipitation procedure
  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 20-50 μl 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, 200 μl lysate (200-500 g 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 1 μl 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 g/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 μl 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