DYKDDDDK Tag (FLAG) Mouse Monoclonal Antibody [A2-A4]
cat.: M1403-2
Product Type: Mouse monoclonal IgG1, primary antibodies
Species reactivity: Species independent
Applications: WB, IP, ELISA, IF-Cell
Clonality: Monoclonal
Clone number: A2-A4
Form: Liquid
Storage condition: Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.
Storage buffer: 1*PBS (pH7.4), 0.2% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 2ug/ul
Purification: Protein G affinity purified.
Isotype: IgG1
Immunogen: Synthetic peptide immune sequence is N-DYKDDDDK-C.
Positive control: DYKDDDDK Tag recombinant protein, C-terminal FLAG-tagged recombinant protein, N-terminal FLAG-tagged recombinant protein.
Recommended Dilutions:
  WB
  IP
  IF-Cell
1:20,000-1:50,000
2-5 µg/ml.
1:500-1:10,000
Alternative names: DDDDK epitope tag DDDK ddk DYKDDDDK DYKDDDDK epitope tag DYKDDDDK tag ECS epitope tag ECS tag Enterokinase Cleavage Site epitope tag Enterokinase Cleavage Site tag FLAG FLAG tag
Images
M1403-2_1.jpg Fig1: Western blot analysis of DYKDDDDK Tag on Flag-tagged recombinant protein Flag-Ki67 and Ki67-Flag at different cell lysates level.

Predicted band size: 35 kDa;
Observed band size: 35 kDa;
Exposure time: 60 seconds;
SDS-PAGE gel concentration:12%

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (M1403-2, 1/20,000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/200,000 dilution was used for 1 hour at room temperature.
M1403-2_2.jpg Fig2: Western blot analysis of DYKDDDDK Tag on Flag-tagged recombinant protein 2xflag-Histone H3.1 at different dilutions of HeLa cells with anti-DYDDDDK antibody (M1403-2).

Lane 1: Non-transfected HeLa cell lysates (1/2000)
Lane 2~4: Transfected HeLa cell lysates (1/2500; 1/5000; 1/10,000)
Predicted band size: 15 kDa;
Observed band size: 18 kDa;
Exposure time: 30 seconds;
SDS-PAGE gel concentration:12%.

Cell lysate at 10ug per lane.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (M1403-2) was used at different dilutions in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/200,000 dilution was used for 1 hour at room temperature.
M1403-2_3.jpg Fig3: Immunoprecipitation analysis of N-Flag in HeLa cells.

Lane 1: HeLa transfected with DYKDDDDK-tagged human Histone H3.1 expression vector whole cell lysate (input).
Lane 2: M1403-2 IP in HeLa transfected with DDDDK-tagged human Histone H3.1 expression vector whole cell lysate.
Lane 3: Mouse IgG (HA1027) instead of M1403-2 in HeLa transfected with DYKDDDDK-tagged human Histone H3.1 expression vector whole cell lysate.

Predicted band size: 19 kDa;
Observed band size: 19 kDa;
Exposure time: 60 seconds;

SDS-PAGE gel concerntration: 15%.

DYKDDDDK Tag was immunoprecipitated from 0.5 mg Hela transfected with human 2x Flag- Histone H3.1 expression vector whole cell lysate with M1403-2 2 ug/mL. Western blot was performed from the immunoprecipitate using M1403-2 at 1/5,000 dilution for 1 hour at room temperature. The secondary antibody for IP detection reagent, Goat anti-Mouse IgG-HRP antibody (HA1006), was used at 1:100,000 dilution.
M1403-2_4.jpg Fig4: Immunoprecipitation analysis of C-Flag in HeLa cells.

Lane 1: HeLa transfected with DYKDDDDK-tagged human Siglec15 expression vector whole cell lysate (input).
Lane 2: M1403-2 IP in HeLa transfected with DDDDK-tagged human Siglec15 expression vector whole cell lysate.
Lane 3: Mouse IgG (HA1027) instead of M1403-2 in HeLa transfected with DYKDDDDK-tagged human Siglec15 expression vector whole cell lysate.

DYKDDDDK Tag was immunoprecipitated from 0.5 mg Hela transfected with human Siglec15 expression vector whole cell lysate with M1403-2 2 ug/mL. Western blot was performed from the immunoprecipitate using M1403-2 at 1/2,000 dilution for 1 hour at room temperature. The secondary antibody for IP detection reagent, Goat anti-Mouse IgG-HRP antibody (HA1006), was used at 1:100,000 dilution.
M1403-2_5.jpg Fig5: Immunofluorescent analysis of 2xFlag tagged Histone H3.1 in HeLa cells.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labeling DYKDDDDK tag with M1403-2 at 1/500 dilution, followed by iFluor™ 488 Goat anti-mouse IgG antibody (HA1125) at 1/1000 dilution (green).
M1403-2_6.jpg Fig6: Immunocytochemistry analysis of HeLa cells labeling DYKDDDDK Tag (FLAG) with Mouse anti-DYKDDDDK Tag (FLAG) antibody (M1403-2) at 1/10,000 dilution.

HeLa cells, transfected with empty control (top, negative) / Flag-tagged Claudin 18.2 (middle, positive) / Flag-tagged Histone H3.1 (bottom, positive) expression vector, respectively, were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-DYKDDDDK Tag (FLAG) antibody (M1403-2) at 1/10,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488, HA1125) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

beta Tubulin (ET1602-4, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) were used as the secondary antibody at 1/1,000 dilution.
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.