DYKDDDDK Tag antibody

Details for Product No. ABIN3181074, Supplier: Log in to see
Antigen
Reactivity
Tag
205
104
43
19
18
11
8
6
4
3
2
1
1
Host
Mouse
180
146
30
25
15
Clonality (Clone)
Monoclonal ()
Conjugate
Un-conjugated
36
21
17
7
7
7
7
7
6
6
6
6
6
6
6
5
4
4
4
4
3
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Application
Immunofluorescence (IF), Immunoprecipitation (IP), Western Blotting (WB)
317
113
109
86
77
45
39
9
9
8
7
6
6
4
4
3
3
2
2
2
1
1
1
1
1
1
Options
Supplier
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Supplier Product No.
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'Independent Validation' Badge
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.

Primary Antibody ABIN3181074
Secondary Antibody goat anti-mouse IgG, HRP-linked (Sigma A9044, lot 034M4761)
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.

Validation Images
Western Blotting validation image for anti-DYKDDDDK Tag antibody (ABIN3181074) Western blot analysis of lysates of HEK-293 cells expressing a DYKDDDDK-tagged protei...
'Independent Validation' Badge
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.

Primary Antibody ABIN3181074
Secondary Antibody goat anti-mouse IgG Dylight488 (ThermoFisher Scientific, 35503, lot KA)
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.
Validation Images
Immunocytochemistry validation image for anti-DYKDDDDK Tag antibody (ABIN3181074) A ABIN3181074 (green) detects a heterologously expressed DYKDDDDK-tagged protein in H...
Sequence DYKDDDDK
Clone 2C5
Isotype IgG1
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.
Antigen
Alternative Name Flag-Tag
Target Type Tag
Application Notes WB 1:5000, IP 1:200, IF 1:2000
Restrictions For Research Use only
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.
Supplier Images
Western Blotting (WB) image for anti-DYKDDDDK Tag antibody (ABIN3181074) anti-DYKDDDDK Tag antibody
Immunoprecipitation (IP) image for anti-DYKDDDDK Tag antibody (ABIN3181074) Immunoprecipitation (IP) analysis: 5ug Flag Mouse IgG1 per ml Lysate, WB 1:5000.
Immunofluorescence (IF) image for anti-DYKDDDDK Tag antibody (ABIN3181074) Immunofluorescence (IF) analysis of 293 cells transfected with a Flag-tag protein,1:2...
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