DYKDDDDK Tag anticorps

N° du produit ABIN3181074

Détail du produit anti-DYKDDDDK Tag anticorps

Antigène: DYKDDDDK Tag

Reactivité: Veuillez nous consulter SVP

Hôte: Souris

Clonalité: Monoclonal

Conjugué: Cet anticorp DYKDDDDK Tag est non-conjugé

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

Séquence: DYKDDDDK

Specificité: The antibody detects C-terminal, internal, and N-terminal Flag-tag fusion proteins.

Attributs du produit: Mouse Monoclonal to Flag-Tag.

Purification: The antibody was affinity-purified from mouse ascites by affinity-chromatography using specific immunogen.

Clone: 2C5

Isotype: IgG1

Information d'application

Indications d'application: WB 1:5000, IP 1:200, IF 1:2000

Restrictions: For Research Use only

Independent Validation (WB)

Validation #102752 (Western Blotting)

by: Developmental Biology, Johann-Friedrich-Blumenbach-Institute for Zoology and Anthropology, Georg-August-University of Göttingen

No.: #102752

Date: 06.04.2018

Antigène: DYKDDDDK Tag

Numéro du lot: 02R690396

Application validée: Western Blotting

Contrôle positif: HEK-293 cells transfected with pcDNA3-FE65-FLAG

Contrôle négative: non-transfected HEK-293 cells

Conclusion:

Passed. ABIN3181074 detects a protein of interest in addition to two further bands.

Validation Images

Western blot analysis of lysates of HEK-293 cells expressing a DYKDDDDK-tagged protein (pcDNA3-FE65-FLAG) or without any heterologous protein expression (HEK-293). The blot membrane was incubated with ABIN3181074 diluted 1:5000 (A) or 1:2000 (B) or with a rabbit anti-Flag tag antibody diluted 1:10000 (C) followed by secondary antibody incubation and chemiluminescence.

Protocole

Anticorps primaire: ABIN3181074

Anticorps secondaire: goat anti-mouse IgG, HRP-linked (Sigma A9044, lot 034M4761)

Full Protocol:

• Grow HEK-293 cells (ATCC, CRL-1573) in DMEM+GlutaMAX (Gibco, 10567-014, lot 1922818) supplemented with fetal bovine serum (Gibco, 10500-064, Lot 08FO477K) and Pen/Strep (Gibco, 15140-122, lot 1924797), at 37°C and 5% CO₂ to 70% confluency.
• Transfect cells with Flag-tagged FE65 expression plasmid (pcDNA3-FE65-FLAG; a kind gift of Dr. Sascha Weggen, University of Düsseldorf) using EndofectinMax (GeneCopoeia) following the manufacturer´s instructions.
• Grow cells for 24h.
• Lyse cells in SDS-sample buffer and denature total cellular lysates for 5min at 95°C. Subsequently separate them on a denaturing 10% SDS-PAGE gel (Laemmli 1970) alongside PageRuler Prestained Protein Ladder (ThermoFisher Scientific, 26616).
• Transfer proteins onto 0.2µm Protran membrane (GE Healthcare, 10600004, A10043108) with a western blotting system for 1h at 100V (Towbin et al., 1979).
• Block the membrane in TBST (50mM Tris-HCl pH7.4, 150mM NaCl, 0.2% Tween 20) containing 5% milk (blocking solution) for 60min at RT.
• Incubate membrane with primary mouse anti-DYKDDDDK antibody (antibodies-online, ABIN3181074, lot 02R690396) diluted 1:2000 in blocking solution ON at 4°C.
• Wash membrane with TBST for 45min at RT.
• Incubate membrane with secondary goat anti-mouse IgG, HRP-linked (Sigma A9044, lot 034M4761) diluted 1:2500 in blocking solution for 45min at RT.
• Wash membrane 6x 5min each with TBST.
• Reveal protein bands using Clarity Max Western ECL substrate (Bio-Rad, 1705062) and capture images via Chemidoc Imaging System (BioRad).
• Incubate a secondary blot membrane with primary rabbit anti-Flag tag antibody (Sigma F7425, lot 086m4803V) diluted 1:10000 in blocking solution ON at 4°C.
• Wash membrane with TBST for 45min at RT.
• Incubate membrane with secondary anti-rabbit IgG (H+L), HRP-linked (Jackson ImmunoResearch, 111-035-003, lot 123450) diluted 1:10000 in blocking solution for 45min at RT.
• Wash membrane with TBST for 45min at RT.
• Reveal protein bands using Clarity Max Western ECL substrate (Bio-Rad, 1705062); image capture via Chemidoc Imaging System (BioRad).

Notes:

ABIN3181074 does not detect proteins when diluted 1:5000. It does however detect a protein in the expected molecular mass range at approximately 97kDa as well as two additional proteins when diluted 1:2000. A rabbit anti-Flag tag antibody used as reference detects a protein in the expected molecular mass range as well two additional proteins.

Independent Validation (ICC)

Validation #103031 (Immunocytochemistry)

by: Developmental Biology, Johann-Friedrich-Blumenbach-Institute for Zoology and Anthropology, Georg-August-University of Göttingen

No.: #103031

Date: 06.04.2018

Antigène: DYKDDDDK Tag

Numéro du lot: 02R690396

Application validée: Immunocytochemistry

Contrôle positif: NIH/3T3 cells transfected with pcDNA3-FE65-FLAG

Contrôle négative: Non-transfected NIH/3T3 cells

Conclusion:

Passed. ABIN3181074 specifically recognizes DYKDDDDK-labeled protein in ICC.

Validation Images

A ABIN3181074 (green) detects a heterologously expressed DYKDDDDK-tagged protein in HEK-293 cells (pcDNA3-FE65-FLAG) but does not reveal an antigen in untransfected cells (untransfected). The pictures on the right show the green channel merged with DAPI counterstain (blue). B A different anti-DYKDDDDK tag antibody (red) was used as control for ABIN3181074 (green). The pictures on the right show the red and green channels merged with DAPI counterstain (blue).

Protocole

Anticorps primaire: ABIN3181074

Anticorps secondaire: goat anti-mouse IgG Dylight488 (ThermoFisher Scientific, 35503, lot KA)

Full Protocol:

• Grow NIH/3T3 cells (ATCC, CRL-1658) in DMEM+GlutaMAX (Gibco, 10567-014, Lot 1922818) supplemented with fetal bovine serum (Gibco, 10500-064, lot 08FO477K) and Pen/Strep (Gibco, 15140-122, Lot 1924797), at 37°C and 5% CO₂ to 70% confluency.
• Transfect cells with DYKDDDDK-tagged FE65 expression plasmid (pcDNA3-FE65-FLAG; a kind gift of Dr. Sascha Weggen, University of Düsseldorf) using EndofectinMax (GeneCopoeia) following the manufacturer´s instructions.
• Grow cells for 24h.
• Fix cells in 3.7% paraformaldehyde (in PBS) for 20min at 4°C followed by incubation in 0.3% Triton X-100 for 10min at 4°C.
• Block cells in PBS containing 1% bovine serum albumin and 0.5% Tween-20 (PBT) for 1h at RT.
• Incubate cells with primary
• mouse anti- DYKDDDDK antibody (antibodies-online, ABIN3181074, lot 02R690396) diluted 1:2000 in PBS ON at 4°C or
  • rabbit anti-DYKDDDDK tag antibody (Sigma, F7425, Lot 086m4803V) diluted 1:2000 in PBS ON at 4°C.
• Wash cells with TBST (50mM Tris-HCl pH7.4, 150mM NaCl, 0.1% Tween 20) for 15min at RT.
• Incubate cells with secondary
• goat anti-mouse IgG Dylight488 (ThermoFisher Scientific, 35503, lot KA) diluted 1:500 in PBS for 1h at 37°C or
  • goat anti-rabbit IgG MFP590 (Mobitec, MFP-A1037, lot 3002088) diluted 1:500 in PBS for 1h at 37°C.
• Counterstain DNA with DAPI (Sigma, D9542).
• Image acquisition on Zeiss LSM 510 confocal microscope and processing using Adobe Photoshop 5.0.

Stockage

Format: Liquid

Concentration: 1 mg/mL

Buffer: Liquid in PBS containing 50 % glycerol, 0.5 % BSA and 0.02 % sodium azide.

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.

Conseil sur la manipulation: Avoid repeated freeze/thaw cycles.

Stock: -20 °C

Stockage commentaire: Store at -20°C.

Détails sur DYKDDDDK Tag

Antigène: DYKDDDDK Tag

Autre désignation: Flag-Tag (DYKDDDDK Tag Produits)

Classe de substances: Tag