HA601167

DYKDDDDK Tag ( FLAG ) Recombinant Mouse Monoclonal Antibody [A2-A4-R]

cat.: HA601167

Product Type:	Recombinant Mouse monoclonal IgG1, primary antibodies
Species reactivity:	Species independent
Applications:	WB, IP, IF-Cell, IHC-P
Clonality:	Monoclonal
Clone number:	A2-A4-R

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer:	PBS (pH7.4), 0.1% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Isotype:	IgG1
Immunogen:	Synthetic peptide immune sequence is N-DYKDDDDK-C.
Recommended Dilutions: WB IP IF-Cell IHC-P	1:5,000-1:10,000 2-5 µg/ml. 1:250-1:10,000 1:1,000
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

Fig1: Western blot analysis of DYKDDDDK Tag ( FLAG ) on different lysates with Mouse anti-DYKDDDDK Tag ( FLAG ) antibody (HA601167) at 1/2,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa transfected with FLAG-tagged Histon H3.1 (N-terminal) cell lysate

Lysates/proteins at 5 µg/Lane.

Predicted band size: 15 kDa
Observed band size: 18 kDa

Exposure time: 1 minute 30 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA601167) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.

Fig2: Western blot analysis of DYKDDDDK Tag ( FLAG ) on different lysates with Mouse anti-DYKDDDDK Tag ( FLAG ) antibody (HA601167) at 1/2,000 dilution.

Lane 1: L-929 cell lysate
Lane 2: L-929 transfected with FLAG-tagged CD5 (C-terminal) cell lysate

Lysates/proteins at 5 µg/Lane.

Predicted band size: 65 kDa
Observed band size: 65 kDa

Exposure time: 1 minute 59 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA601167) at 1/2,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.

	Fig3: DYKDDDDK Tag ( FLAG ) was immunoprecipitated in 2µg HeLa transfected with FLAG-tagged Histon H3.1 (N-terminal) cell lysate with HA601167. Western blot was performed from the immunoprecipitate using DYKDDDDK Tag(FLAG) (M1403-2) at 1/1,000 dilution. Anti-Mouse IgG for IP Nano-Secondary Antibody (NBI02H) at 1:5,000 dilution was used for 1 hour at room temperature. Lane 1: HeLa transfected with FLAG-tagged Histon H3.1 (N-terminal) cell lysate (input). Lane 2: Mouse IgG instead of HA601167 IP in HeLa transfected with FLAG-tagged Histon H3.1 (N-terminal) cell lysate. Lane 3: HA601167 IP in HeLa transfected with FLAG-tagged Histon H3.1 (N-terminal) cell lysate. Blocking/Dilution buffer: 5% NFDM/TBST.
	Fig4: DYKDDDDK Tag ( FLAG ) was immunoprecipitated in 2µg L-929 transfected with FLAG-tagged CD5 (C-terminal) cell lysate with HA601167. Western blot was performed from the immunoprecipitate using DYKDDDDK Tag(FLAG) (M1403-2) at 1/1,000 dilution. Anti-Mouse IgG for IP Nano-Secondary Antibody (NBI02H) at 1:5,000 dilution was used for 1 hour at room temperature. Lane 1: L-929 transfected with FLAG-tagged CD5 (C-terminal) cell lysate (input). Lane 2: Mouse IgG instead of HA601167 IP in L-929 transfected with FLAG-tagged CD5 (C-terminal) cell lysate. Lane 3: HA601167 IP in L-929 transfected with FLAG-tagged CD5 (C-terminal) cell lysate. Blocking/Dilution buffer: 5% NFDM/TBST.
	Fig5: Immunohistochemical analysis of paraffin-embedded HeLa transfected with FLAG-tagged Histon H3.1 (N-terminal) cells with Mouse anti-DYKDDDDK Tag ( FLAG ) antibody (HA601167) at 1/1,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA601167) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Fig6: Immunohistochemical analysis of paraffin-embedded HeLa transfected with FLAG-tagged Claudin 18.2 (C-terminal) cells with Mouse anti-DYKDDDDK Tag ( FLAG ) antibody (HA601167) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA601167) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Fig7: Immunocytochemistry analysis of HeLa cells labeling DYKDDDDK Tag ( FLAG ) with Mouse anti-DYKDDDDK Tag ( FLAG ) antibody (HA601167) at 1/250 dilution.

HeLa cells, transfected with FLAG-tagged empty control, Claudin 18.2 (C-terminal) or Histone H3.1 (N-terminal) expression vector, respectively, were fixed in 4% paraformaldehyde for 10 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 (HA601167) at 1/250 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.

Fig8: Immunocytochemistry analysis of HeLa cells labeling DYKDDDDK Tag ( FLAG ) with Mouse anti-DYKDDDDK Tag ( FLAG ) antibody (HA601167) at 1/10,000 dilution.

HeLa cells, transfected with FLAG-tagged empty control, Claudin 18.2 (C-terminal) or Histone H3.1 (N-terminal) expression vector, respectively, were fixed in 4% paraformaldehyde for 10 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 (HA601167) 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”.