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DYKDDDDK Fab-Trap Agarose

Description
The DYKDDDDK Fab-Trap™ Agarose is a premium affinity resin 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.

Specificity
The DYKDDDDK Fab-Trap™ Agarose specifically binds to 1x and 3xDYKDDDDK-tag sequence (1x and 3xFlag®-tag). It binds to same epitope as Sigma's Anti-FLAG® M2 Antibody and is compatible with N-, C-terminal or internal tagging.

Applications
Immunoprecipitation (IP) / Co-IP
Mass spectrometry (MS)
Protein purification
ChIP/ RIP analysis
On-bead enzyme assays

Try out the DYKDDDDK IP Starter Pack (ffap)
DYKDDDDK Fab-Trap™ Agarose (20 reactions, product code ffa-20) for immunoprecipitation, 3xDYKDDDDK-peptide (1mg, fp-1) for elution and DYKDDDDK tag Polyclonal antibody (Binds to FLAG® tag epitope) (20 µL, 20543-1-AP-20UL) for detection in Western blots.

Product Size Code Price Buy
Product DYKDDDDK Fab-Trap Agarose Size 250 µL (10 reactions) Code ffa-10 Price $ 275
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Product DYKDDDDK Fab-Trap Agarose Size 500 µL (20 reactions) Code ffa-20 Price $ 475
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Product DYKDDDDK Fab-Trap Agarose Size 2.5 mL (100 reactions) Code ffa-100 Price $ 2050
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Product DYKDDDDK Fab-Trap Agarose Size 5 mL (200 reactions) Code ffa-200 Price Please inquire
Product DYKDDDDK Fab-Trap Agarose Size 10 mL (400 reactions) Code ffa-400 Price Please inquire
Product DYKDDDDK Fab-Trap Agarose Kit Size 500 µL (20 reactions) Code ffak-20 Price $ 595
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Product 3xDYKDDDDK-peptide Size 1 mg Code fp-1 Price $ 105
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Product DYKDDDDK Immunoprecipitation Starter Pack Size 20 reactions Code ffap Price $ 540
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Product Binding Control Agarose Beads Size 500 µL (20 reactions) Code bab-20 Price $ 75
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Product Spin Columns Size 10 units Code sct-10 Price $ 35
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Product Spin Columns Size 20 units Code sct-20 Price $ 60
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Product Spin Columns Size 50 units Code sct-50 Price $ 130
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Wash buffer compatibility
0.5 % Nonidet P40 Substitute, 5 % Tween™ 20, 2 M NaCl, 10 mM TCEP, 10 mM DTT, 10 mM ß-mercaptoethanol, 2 M Urea

Specificity
1x and 3xDYKDDDDK-tag sequence (1x and 3xFlag®-tag). The DYKDDDDK Fab-Trap™ Agarose binds to same epitope as Sigma's Anti-FLAG® M2 antibody.

Binding capacity
25 µL slurry bind about 12.5 – 15 µg of recombinant 3xDYKDDDDK-tagged protein (~30 kDa)

Clone
Recombinant Fab fragment of monoclonal mouse IgG1 M2.1

Bead properties
Bead size: ~ 90 µm (cross-linked 4 % agarose beads)
Storage buffer: 1x PBS, Preservative: 0.09 % sodium azide

Elution
- 3xDYKDDDDK-peptide
- SDS sample buffer
- 0.2 M glycine pH 2.5
Please consider whether elution is really needed. We recommend on-bead assays like on-bead digestion for MS analysis.

Compatibility with mass spectrometry
The DYKDDDDK Fab-Trap Agarose is optimized for on-bead digestion. 

How to cite this product
RRID: AB_2894836

Storage instructions
Shipped at ambient temperature. Upon receipt store at 4°C. Stable for one year.

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.

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 Immunoprecipitation Starter Pack

You look for a cost-effective pack for Flag® IP or you are planning your first IP of a Flag-tagged protein? Or you want to test the DYKDDDDK Fab-Trap™ Agarose?

Here is a Starter Pack (ffap) containing

Benefits

  • Pulldown of Flag®-tagged proteins
  • Lowest background in class
  • Less contaminating antibody chains
  • Reliable: recombinantly expressed Fab-fragment derived from M2 clone
  • Small Fab-fragment tolerates harsh wash buffer conditions
  • Convenient and fast: Easy handling and ready-to-use beads
  • Highly selective binding of both N-and C-terminal Flag-tag

3xDYKDDDDK-peptide

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

The 3xDYKDDDDK-peptide  has the amino acid sequence DYKDDDDK DYKDDDDK DYKDDDDK.

DYKDDDDK Fab-Trap Agarose Kit

The DYKDDDDK Fab-Trap Agarose is also available in a kit, including:

  • DYKDDDDK Fab-Trap Agarose
  • Lysis*, dilution and wash buffers

*The lysis buffer provided is suitable for mammalian cells. For other cell types use another suitable lysis buffer.

Spin columns

  • Easy and convenient handling
  • Rapid washing and clean elution of bound proteins
  • Simplify the pulldown of your protein
  • Spin columns for DYKDDDDK Fab-Trap Agarose

Binding Control for Agarose Beads

  • Control for unspecific binding of proteins, DNA, etc. to beads
  • Pre-clearing of cell lysate
  • Binding control for DYKDDDDK Fab-Trap Agarose

Why is the DYKDDDDK Fab-Trap superior?

  1. Minimal contamination by Fab-fragment of the DYKDDDDK Fab-Trap
    The DYKDDDDK Fab-Trap consists of a Fab-fragment, which is covalently bound to beads. In contrast to antibody-based immunoprecipitation reagents, there is no contamination from DYKDDDDK Fab-Trap on your SDS-PAGE and Western blot above 25 kDa, resulting in cleaner SDS-PAGE and Western blots. Moreover, you will find less antibody derived peptide contaminations in subsequent mass spectrometric analysis.
  2. Lowest Background in class
    ChromoTek’s DYKDDDDK Fab-Trap is an anti-Flag affinity reagent based on a Fab-fragment, which is only a third of the size of a conventional antibody. Due to its smaller size, the Fab-fragment minimizes unwanted interaction with the host-cell proteome, resulting in efficient immunoprecipitations with superior purity and less background.
  3. Small Fab-fragment tolerates harsh wash buffer conditions
    The small Fab-fragment of the DYKDDDDK Fab-Trap tolerates harsher washing conditions as comparable anti-Flag antibody affinity resins. The possibility to use harsher wash buffers, enables effective removal of unwanted cellular proteins for highest purity immunoprecipitation.
  4. Tight and highly selective binding of a single Flag-tag
    The DYKDDDDK Fab-Trap binds with high specificity to N- or C-terminal Flag-tagged proteins. In addition, it has a high affinity to both 1x or 3xFlag-tag enabling a broad experimental applicability.
  5. Ready-to-use reagent
    DYKDDDDK Fab-Trap is a ready-to-use reagent that comes pre-conjugated to beads. There is no need for tedious and time-consuming pre-incubation steps required for pulldowns with protein A or G beads making the immunoprecipitation faster, more convenient, and easier to reproduce.
  6. Mild, competitive elution with DYKDDDDK-peptide or acidic conditions
    DYKDDDDK Fab-Trap can be mildly eluted with 3xDYKDDDDK-peptide, preserving the native structure of your Flag-tagged protein for structural and activity downstream analysis. In addition, convenient elution by low pH or SDS-sample buffer offers fast and efficient elution performance.
  7. Reproducible results
    DYKDDDDK Fab-Trap binds to the same epitope as Sigma’s ANTI-FLAG® M2 antibody. The Fab-fragment of the DYKDDDDK Fab-Trap is recombinantly produced for the generation of highly reliable data.

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