DYKDDDDK Tag antibody

Catalog No. ABIN559688

Product Details anti-DYKDDDDK Tag Antibody

Target: DYKDDDDK Tag

Reactivity: Please inquire

Host: Mouse

Clonality: Monoclonal

Conjugate: This DYKDDDDK Tag antibody is un-conjugated

Application: Western Blotting (WB), ELISA, Immunoprecipitation (IP), Dot Blot (DB), Immunostaining (ISt)

Sequence: DYKDDDDK

Specificity: Recognizes the N-terminal, C-terminal or internal DYKDDDDK-tagged fusion proteins

Sensitivity: WB (with ECL): 1-5ng of DYKDDDDK-tagged fusion proteins

Purification: Protein A affinity chromatography from mouse ascites fluid

Immunogen: DYKDDDDK (DYKDDDDK) synthetic peptide conjugated to KLH

Clone: FG4R

Isotype: IgG1

Application Details

Application Notes: WB (with ECL): 1:1000-3000 dilution (incubate for one hour at room temperature) / IS: 1:500-2000 dilution / For best results with other assays (e.g.: Dot, ELISA, IP, etc), please determine optimal working dilution by titration test

Restrictions: For Research Use only

Independent Validation (IF)

Validation #102796 (Immunofluorescence)

by: Martinelli lab, Neuroscience Department, UConn Health

No.: #102796

Date: 02/13/2018

Antigen: DYKDDDDK-tag

Lot Number: 02017

Method validated: Immunofluorescence

Positive Control: African green monkey kidney fibroblast-like COS7 cells transfected with BAI3, a transmembrane protein with a surface presented DYKDDDDK-tag

Negative Control: untransfected COS7 cells

Notes:

Passed. Signal was observed as expected. There was never any signal on untransfected cells, demonstrating specificity of the antibody.

Validation Images

COS7 cells were transfected to express a DYKDDDDK-tagged and surface presented BAI3 protein. Indirect immunoflouresence using ABIN1715174 reveals specific signal for the DYKDDDDK-tag (middle) on the cell surface. No colocalization of the ABIN1715174 and nuclear stain (DAPI, left) was observed (merge, right). No signal was ever observed in any untransfected cell.

Full Methods

Primary Antibody: ABIN1715174

Secondary Antibody: goat anti-mouse AF488 conjugated antibody (Life Technologies, A11029, lot 1829920)

Full Protocol:

• Grow COS7 cells in DMEM (Biological Industries, 01-052-1A, lot 1728619) supplemented with FBS (Hyclone, SH3039602) without antibiotics, at 37°C and 5% CO₂ in 1ml on glass coverslips on 24-well plate.
• Transfect cells with calcium phosphate method with a eukaryotic expression construct to express a DYKDDDDK-tagged BAI3, which will be surface presented.
• Grow cells for 24h.
• Fix cells on coverslips in 4% PFA for 15min at 4°C.
• Wash cells 3x for 5min with PBS.
• Block non-specific binding with 5% goat serum in PBS for 1h at RT.
• Incubate cells with primary mouse anti-DYKDDDDK-tag antibody (antibodies-online, ABIN1715174, lot 02017) diluted 1:2000 in blocking solution ON at 4°C.
• Wash cells 3x for 5min with PBS.
• Incubate cells with secondary goat anti-mouse AF488 conjugated antibody (Life Technologies, A11029, lot 1829920) diluted 1:400 in blocking buffer for 1h at RT protected from light.
• Wash cells 3x for 5min with PBS.
• Mount coverslips on glass slides in Fluoromount-G with DAPI mounting medium (ThermoFisher Scientific, 00-4959-52, lot B2215-N915).
• Image acquisition on a Zeiss Axiovert epifluorescence with with a 40x objective, using a Zeiss ApoTome for optical sectioning.

Handling

Concentration: 1mg/mL

Buffer: 10mM PBS, pH 7.2, 0.05% NaN3

Preservative: Sodium azide

Precaution of Use: This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: -20 °C

Target Details for DYKDDDDK Tag

Target: DYKDDDDK Tag

Alternative Name: FLAG Tag (DYKDDDDK Tag Products)

Target Type: Tag