DYKDDDDK Tag antibody

Catalog No. ABIN1112984

Product Details anti-DYKDDDDK Tag Antibody

Target: DYKDDDDK Tag

Reactivity: Please inquire

Host: Mouse

Conjugate: This DYKDDDDK Tag antibody is un-conjugated

Application: Western Blotting (WB), Immunoprecipitation (IP), Immunofluorescence (IF)

Sequence: DYKDDDDK

Immunogen: A synthetic peptide DYKDDDDK conjugated to KLH.

Isotype: IgG1

Application Details

Application Notes: WB (1:1000-10000), IP (1:100-500), IF (1:200-1000). Other applications have not been tested. The optimal dilutions should be determined by end user.

Restrictions: For Research Use only

Independent Validation (IF)

Validation #028889 (Immunofluorescence)

by: Reveal Biosciences

No.: #028889

Date: 11/07/2013

Lot Number: L21/2013G

Method validated: Immunofluorescence

Positive Control: HEK293 cells transfected with DYKDDDDK Tag

Negative Control: Untransfected HEK293 cells

Notes: Signal was detected in positive control samples but not in negative control samples.

Validation Images

Figure 1: : Immunofluorescence analysis of cultured cells using DYKDDDDK Tag antibody (Catalog number ABIN1112984, Lot number L21/2013G). Flag is present in the positive control (HEK293 cells transfected with DYKDDDDK Tag panel 1, transfected cells are indicated by green fluorescent staining) and absent from the negative control (untransfected HEK293 cells panel 2). No staining was observed in the isotype (panel 3) and secondary only (panel 4) controls. DAPI was used as a nuclear counterstain (indicated by blue fluorescent staining). Scale bar = 25 µm.

Full Methods

Primary Antibody:

• Antibody: DYKDDDDK Tag antibody
• Catalog number: ABIN1112984
• Lot number: L21/2013G

Secondary Antibody:

• Antibody: Goat anti-mouse IgG (H+L) IF488 conjugated antibody
• Lot number: S6/2013S

Full Protocol:

• HEK293 cells were grown directly on coverslips and transfected with a DYKDDDDK tagged protein.
• Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature (RT).
• Fixed cells were rinsed three times in PBS for 5 min each at RT.
• Cells were blocked in 1 X PBS / 5% normal goat serum / 0.3% Triton X-100 for 20 min at RT.
• Cells were incubated with primary antibody diluted 1:100 in Universal Antibody Dilution Buffer (Electron
• Microscopy Sciences, 25885-05) overnight at 4°C.
• Cells were rinsed three times in PBS for 5 min each at RT.
• Cells were incubated with secondary antibody diluted 1:150 in Universal Antibody Dilution Buffer
• (Electron Microscopy Sciences, 25885-05) for 60 min at RT in dark.
• Cells were rinsed three times in PBS for 5 min each at RT.
• Coverslips were mounted on slides with Fluoro-Gel II with DAPI (Electron Microscopy Sciences Cat. No: 17985-50).
• Stained cells were imaged with an EIDAQ 100 fluorescent microscope.

Experimental Notes: None

Handling

Format: Liquid

Buffer: PBS (pH7.4), 50% Glycerol and 0.02% Sodium Azide.

Preservative: Sodium azide, Thimerosal (Merthiolate)

Storage: 4 °C

Storage Comment: Store at 2-8 °C for 1 month. Or aliquot and store at -20 °C or below for long term. Avoid repeated freeze and thaw cycles.

Target Details for DYKDDDDK Tag

Target: DYKDDDDK Tag

Alternative Name: Flag Tag (DYKDDDDK Tag Products)

Target Type: Tag

Background: DYKDDDDK-Tag, or DYKDDDDK octapeptide, is a polypeptide protein tag that can be added to a protein using recombinant DNA technology. It can be used for affinity chromatography, then used to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism. The peptide sequence of the DYKDDDDK-Tag is: N-DYKDDDDK-C (1012 Da). It can be used in conjunction with other affinity tags, for example a polyhistidine tag (His-tag), HA-tag or myc-tag. It can be fused to the C-terminus or the N-terminus of a protein.