Anti-DYKDDDDK Agarose

Catalog No.	Product Name	Size	List Price (US$)	Quantity
PA000517-M20018S	Biotinylated Recombinant Anti-DYKDDDDK-Tag Monoclonal Antibody (Clone L1.1), Human IgG1 Fc Silent Kappa (Agarose conjugated)	500 ul	216.00
PA000517-M20018L	Biotinylated Recombinant Anti-DYKDDDDK-Tag Monoclonal Antibody (Clone L1.1), Human IgG1 Fc Silent Kappa (Agarose conjugated)	5 ml	960.00

Catalog No.

Product Name

Size

List Price (US$)

Quantity

PA000517-M20018S

Biotinylated Recombinant Anti-DYKDDDDK-Tag Monoclonal Antibody (Clone L1.1), Human IgG1 Fc Silent Kappa (Agarose conjugated)

500 ul

216.00

PA000517-M20018L

Biotinylated Recombinant Anti-DYKDDDDK-Tag Monoclonal Antibody (Clone L1.1), Human IgG1 Fc Silent Kappa (Agarose conjugated)

5 ml

960.00

Description

PA000517-M20018: Biotinylated Recombinant Anti-DYKDDDDK-Tag Monoclonal Antibody (Clone L1.1), Human IgG1 Fc Silent Kappa (conjugated to Agarose)

DYKDDDDK-Tag mouse monoclonal antibody conjugated to Agarose is made by covalently linking a monoclonal anti-DYKDDDDK antibody to the agarose which enables immunoprecipitation (IP) of DYKDDDDK tagged proteins or co-immunoprecipitation (Co-IP) of their interacting partners.
Source: This monoclonal antibody is produced by immunizing animals with a synthetic peptide containing epitope DYKDDDDK (KLH-coupled).
Clone: L1.1.
Isotype: Human IgG1 Fc Silent Kappa.
Specificity: Anti-DYKDDDDK-Tag Mouse mAb detects transfected proteins containing the DYKDDDDK epitope tag.
Storage: The product is supplied as a 50% slurry in storage buffer (1xPBS, pH 7.4, containing 0.1% NaN3). Store the product at 4°C and do not freeze.
RECOMMENDED ELUTION BUFFER: 0.2 M Glycine, pH 2.5.

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 ul 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 ul lysate (200-500 ug 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 2,000xg, 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 ul 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 ul of 1.5 M Tris, pH 9.0 per 20 ul Elution buffer.

Option 2. Elution with DYKDDDDK peptide
Add 30-50 ul DYKDDDDK peptide solution (100 ug/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 ul 2x 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.