DYKDDDDK IP Starter Pack

Description
The DYKDDDDK Immunoprecipitation Starter Pack is a premium solution for efficient immunoprecipitation (IP) of Flag®-tagged proteins, competitive peptide elution, and sensitive Western blot (WB) detection.

Specificity
1x and 3xDYKDDDDK-tag sequence (1x and 3xFlag®-tag). Compatible with N-, C- terminal or internal tagging.

Applications
Immunoprecipitation (IP) / Co-IP

Components:

Product Size Code Price Buy
Product DYKDDDDK Immunoprecipitation Starter Pack Size 20 reactions Code ffap Price $ 540
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What is the Fab-fragment of the DYKDDDDK Fab-Trap™ based on?

The DYKDDDDK Fab-Trap™ consists of a Fab-fragment derived from the mouse IgG1 clone M2, which is covalently bound to Agarose beads. It recognizes the same epitope as the M2 antibody. Due to its small size, the Fab-fragment has several advantages for IP, such as a lower background and broader wash buffer compatibility.The DYKDDDDK Fab-Trap™ consists of a Fab-fragment derived from the mouse IgG1 clone M2, which is covalently bound to Agarose beads. It recognizes the same epitope as the M2 antibody. Due to its small size, the Fab-fragment has several advantages for IP, such as a lower background and broader wash buffer compatibility.

How can I avoid that the Fab-fragment is released from the beads during elution and becomes visible on SDS-PAGE or WB?

The Fab-fragment is released during elution with SDS-sample buffer and is visible on SDS-PAGE or WB at ~25 kDa. To avoid release of the Fab-fragment, you may use milder elution options such as elution with low pH or peptide elution with the 3xDYKDDDDK-peptide

Does the DYKDDDDK Fab-Trap™ bind to 1x and 3xFlag®-tag?

DYKDDDDK Fab-Trap™ binds nicely to both, the 1x and 3xFlag®-tag. Elution of 1x and 3xFlag®-tagged proteins should be done with the 3xDYKDDDDK-peptide

Should I use an N-terminal or C-terminal fusion tag? Can I also tag my protein internally?

Both N-terminal or C-terminal fusion tags work well with the DYKDDDDK Fab-Trap™. The compatibility of an internal DYKDDDDK-tag depends on the fusion protein and must be tested case by case.

What is the affinity (Kd) of the DYKDDDDK Fab-Trap™?

The dissociation constant Kd of the Fab-fragment of the DYKDDDDK Fab-Trap™ to a Flag®-tagged fusion protein is 36 nM.

Do I need to conjugate the DYKDDDDK Fab-Trap™ to protein A or G beads?

No, the DYKDDDDK Fab-Trap™ is a ready-to-use reagent. It comes pre-conjugated to Agarose beads for convenient and time efficient immunoprecipitation.

How can I detect the 1x or 3xFlag®-tagged protein in Western blotting?

For Western blot we recommend Proteintech’s DYKDDDDK tag Polyclonal antibody (Binds to FLAG® tag epitope)  (cat # 20543-1-AP) from rabbit in combination with an anti-rabbit secondary. Alternatively, an anti-Flag® antibody pre-conjugated to HRP can be used. 
It is not recommended to use an anti-Flag®-tag antibody from mouse (e.g., clone M2) in combination with an anti-mouse secondary. The anti-mouse secondary can recognize both the primary mouse anti-Flag®-tag antibody and the Fab-fragment of the DYKDDDDK Fab-Trap™, which is released from the beads during elution with SDS-sample buffer and has a size around 25 kDa.
 

What is the amino acid sequence of the 1x and 3xFlag®-tag?

The amino acid sequence of 1xFlag®-tag is DYKDDDDK. The amino acid sequence of the 3x-Flag®-tag is DYKDHD-G-DYKDHD-I-DYKDDDDK. Note that the sequence of the 3xFlag®-tag is an optimized peptide sequence. You can find more information about the DYKDDDDK-tag in our blog: Flag-tag and 3x Flag-tag: An epitope tag for capture and detection experiments.

How can I avoid unspecific protein interactions binding to the DYKDDDDK Fab-Trap™?

For preclearing of your sample, we recommend using our binding controls (bab-20).
Preclearing with an isotype control is not necessary as the DYKDDDDK Fab-Trap™ is only 50 kDa in size and lacks the Fc domain which minimizes unwanted interactions with the host cell proteome.
Please find more information in our Troubleshooting guide.
 

Do I need to elute my protein of interest from the beads for enzymatic assays?

No, you can directly perform your enzymatic assay on the beads if the active center is not blocked.
Please find more information in our application note "enzymatic activity assay" .
 

DYKDDDDK Fab-Trap Agarose

The DYKDDDDK Fab-Trap Agarose (20 reactions, product code ffa-20) is a premium reagent for efficient immunoprecipitation (IP) of Flag-tagged proteins. The DYKDDDDK Fab-Trap Agarose consists of a Fab-fragment, which is covalently bound to agarose beads.

3xDYKDDDDK-peptide

The 3xDYKDDDDK-peptide (1mg, fp-1) may be used for the elution of native and functional Flag-tagged proteins bound to DYKDDDDK Fab-Trap or anti-DYKDDDDK antibodies.

The amino acid sequence is MDYKDHDGDYKDHDIDYKDDDDK.

DYKDDDDK tag Polyclonal antibody (Binds to FLAG® tag epitope)

DYKDDDDK tag Polyclonal antibody (Binds to FLAG® tag epitope) (20 µL, 20543-1-AP-20UL) for detection in Western blots.

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Only for research applications, not for diagnostic or therapeutic use!