DYKDDDDK Tag anticorps

N° du produit ABIN559688

L’anticorps Souris Monoclonal anti-DYKDDDDK Tag a été validé pour WB, ELISA, IP, DB et ISt et a été validé indépendamment. Il convient pour détecter DYKDDDDK Tag dans des échantillons de .

Aperçu rapide pour DYKDDDDK Tag anticorps (ABIN559688)

Antigène: DYKDDDDK Tag

Reactivité: Veuillez nous consulter SVP

Hôte: Souris

Clonalité: Monoclonal

Conjugué: Cet anticorp DYKDDDDK Tag est non-conjugé

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

Clone: FG4R

Détail du produit anti-DYKDDDDK Tag anticorps

Séquence: DYKDDDDK

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

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

Purification: Protein A affinity chromatography from mouse ascites fluid

Immunogène: DYKDDDDK (DYKDDDDK) synthetic peptide conjugated to KLH

Isotype: IgG1

Information d'application

Indications d'application: 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: 13.02.2018

Antigène: DYKDDDDK-tag

Numéro du lot: 02017

Application validée: Immunofluorescence

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

Contrôle négative: untransfected COS7 cells

Conclusion:

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.

Protocole

Anticorps primaire: ABIN1715174

Anticorps secondaire: 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.

Stockage

Concentration: 1mg/mL

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

Agent conservateur: Sodium azide

Précaution d'utilisation: This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Stock: -20 °C

Détails sur DYKDDDDK Tag

Antigène: DYKDDDDK Tag

Autre désignation: FLAG Tag

Classe de substances: Tag