DYKDDDDK Tag antibody

Catalog No. ABIN3178554

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), Immunoprecipitation (IP), Immunofluorescence (IF)

Sequence: DYKDDDDK

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

Characteristics: Mouse Monoclonal to Flag-Tag.

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

Clone: 2C5

Isotype: IgG1

Application Details

Application Notes: 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: 04/06/2018

Antigen: DYKDDDDK Tag

Lot Number: 02R690396

Method validated: Western Blotting

Positive Control: HEK-293 cells transfected with pcDNA3-FE65-FLAG

Negative Control: non-transfected HEK-293 cells

Notes:

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.

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).

Full Methods

Primary Antibody: ABIN3181074

Secondary Antibody: 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).

Experimental 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: 04/06/2018

Antigen: DYKDDDDK Tag

Lot Number: 02R690396

Method validated: Immunocytochemistry

Positive Control: NIH/3T3 cells transfected with pcDNA3-FE65-FLAG

Negative Control: Non-transfected NIH/3T3 cells

Notes:

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

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.

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).

Full Methods

Primary Antibody: ABIN3181074

Secondary Antibody: 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.

Handling

Format: Liquid

Concentration: 1 mg/mL

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

Preservative: Sodium azide

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

Handling Advice: Avoid repeated freeze/thaw cycles.

Storage: -20 °C

Storage Comment: Store at -20°C.

Target Details for DYKDDDDK Tag

Target: DYKDDDDK Tag

Alternative Name: Flag-Tag (DYKDDDDK Tag Products)

Target Type: Tag