HAK21016

Epitope Tag Antibody Sampler Kit

cat.: HAK21016

Product Type:	Antibody Sampler Kit
Species reactivity:	Species independent
Applications:	WB, IF-Cell, IP

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer:	1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Images

Fig1: Western blot analysis of GST on different lysates with Rabbit anti-GST antibody (ET1611-47) at 1/50,000 dilution.

Lane 1: 293T transfected with GST cell lysate (2.5 µg/Lane)
Lane 2: 293T transfected with GST-tagged Histone H3.1 cell lysate (15 µg/Lane)

Exposure time: 46 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 (ET1611-47) at 1/50,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Fig2: GST was immunoprecipitated in 0.2mg 293T-OE-GST cell lysate with ET1611-47 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1611-47 at 1/50,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: 293T transfected with GST-tagged Histone H3.1 cell lysate (input)
Lane 2: Rabbit IgG instead of ET1611-47 in 293T transfected with GST-tagged Histone H3.1 cell lysate
Lane 3: ET1611-47 IP in 293T transfected with GST-tagged Histone H3.1 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 24 seconds

Fig3: Immunocytochemistry analysis of HeLa transfected with GST-tagged Histone H3.1 cells labeling GST with Rabbit anti-GST antibody (ET1611-47) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 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 Rabbit anti-GST antibody (ET1611-47) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) 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.

Histone H3 (HA601278, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Fig4: Western blot analysis of Myc tag on different lysates with Mouse anti-Myc tag antibody (HA601081) at different dilutions.

Lane 1/2: C-terminal Myc-tag fusion protein lysate
Lane 3/4: N-terminal Myc-tag fusion protein lysate

Lysates/proteins at 50 ng/Lane.

Observed band size: 34 kDa

Exposure time: 30 seconds;

12% 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 (HA601081) at different dilutions was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:100,000 dilution was used for 1 hour at room temperature.

Fig5: Myc-tag was immunoprecipitated in 2µg C-terminal Myc-tag fusion protein lysate with HA601081. Western blot was performed from the immunoprecipitate using HA601081 at 1/2,000 dilution. Anti-Mouse IgG for IP Nano-secondary antibody (NBI02H) at 1:5,000 dilution was used for 60 mins at room temperature.

Lane 1: C-terminal Myc-tag fusion protein lysate (input).
Lane 2: HA601081 IP in C-terminal Myc-tag fusion protein lysate.
Lane 3: Mouse IgG instead of HA601081 IP in C-terminal Myc-tag fusion protein lysate.

Blocking/Dilution buffer: 5% NFDM/TBST.

Fig6: Immunocytochemistry analysis of 293T cells labeling Myc tag with Mouse anti-Myc tag antibody (HA601081) at 1/1,000 dilution.

293T cells, transfected with Myc-tagged empty control, Claudin18.2 (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-Myc tag antibody (HA601081) at 1/1,000 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.

Myc Tag (R1208-1, red) was stained at 1/200 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) was used as the secondary antibody at 1/1,000 dilution.

Fig7: Western blot analysis of HA-tag on different lysates with Mouse anti-HA-tag antibody (HA721750) at 1/5,000 dilution.

Lane 1: 293T cell lysate
Lane 2: 293T transfected with HA-tagged Nanos homolog 3 (N-terminal) cell lysate
Lane 3: 293T transfected with HA-tagged LIPT1 (N-terminal) cell lysate
Lane 4: 293T transfected with HA-tagged CXCL13 (C-terminal) cell lysate
Lane 5: 293T transfected with HA-tagged CLK4 (C-terminal) cell lysate

Lysates/proteins at 10 µg/Lane.

Exposure time: 3 minutes;

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 (HA721750) at 1/5,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Fig8: Immunocytochemistry analysis of 293T cells labeling HA tag with Rabbit anti-HA tag antibody (HA721750) at 1/1,000 dilution.

293T cells, transfected with HA-tagged empty control, Nanos homolog 3 (N-terminal) or CLK4 (C-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 Rabbit anti-HA tag antibody (HA721750) at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) 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 (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Fig9: Western blot analysis of 6X His tag on different lysates with Rabbit anti-6X His tag antibody (HA722798) at 1/5,000 dilution.

Lane 1: 293T transfected with His-tagged empty control cell lysate
Lane 2: 293T transfected with His-tagged ACAT2 (N-terminal) cell lysate
Lane 3: 293T transfected with His-tagged Histone H3.1 (C-terminal) cell lysate

Lysates/proteins at 10 µg/Lane.

Exposure time: 2 seconds; ECL: K1801;

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 (HA722798) at 1/50,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Fig10: 6X His tag was immunoprecipitated from 0.2 mg 293T transfected with His-tagged Histon H3.1 (C-terminal) cell lysate with HA722798 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA722798 at 1/500 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: 293T transfected with His-tagged Histon H3.1 (C-terminal) cell lysate (input)
Lane 2: HA722798 IP in 293T transfected with His-tagged Histon H3.1 (C-terminal) cell lysate
Lane 3: Rabbit IgG instead of HA722798 in 293T transfected with His-tagged Histon H3.1 (C-terminal) cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 8 seconds; ECL: K1801

Fig11: Immunocytochemistry analysis of HeLa cells labeling 6X His tag with Rabbit anti-6X His tag antibody (HA722798) at 1/5,000 dilution.

HeLa cells, transfected with His-tagged Histone H3.1 (C-terminal) or ACAT2 (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-6X His tag antibody (HA722798) at 1/50,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) 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 (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Fig12: Immunohistochemical analysis of paraffin-embedded HeLa transfected with His-tagged Histon H3.1 (C-terminal) cells with Rabbit anti-6X His tag antibody (HA722798) at 1/5,000 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 (HA722798) 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.

Fig13: Western blot analysis of DYKDDDDK Tag (FLAG) on different lysates with Rabbit anti-DYKDDDDK Tag (FLAG) antibody (HA722780) at 1/5,000 dilution.

Lane 1: 293T transfected with FLAG-tagged empty control cell lysate
Lane 2: 293T transfected with FLAG-tagged MYRF (N-terminal) cell lysate
Lane 3: 293T transfected with FLAG-tagged PAF1 (C-terminal) cell lysate
Lane 4: 293T transfected with FLAG-tagged Histone H3 (C-terminal) cell lysate

Lysates/proteins at 10 µg/Lane.

Exposure time: 2 seconds; ECL: K1801;

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 (HA722780) at 1/5,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Fig14: Immunocytochemistry analysis of HeLa cells labeling DYKDDDDK Tag (FLAG) with Rabbit anti-DYKDDDDK Tag (FLAG) antibody (HA722780) 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 (HA722780) at 1/10,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) 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 (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

	Fig15: DYKDDDDK Tag (FLAG) was immunoprecipitated in 2µg L-929 transfected with FLAG-tagged CD5 (C-terminal) cell lysate with HA722780. Western blot was performed from the immunoprecipitate using DYKDDDDK Tag (FLAG) (HA722780) at 1/1,000 dilution. Mouse anti Rabbit IgG heavy chain (Fc) secondary antibody (M1003-7) at 1/100,000 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: Rabbit IgG instead of HA722780 IP in L-929 transfected with FLAG-tagged CD5 (C-terminal) cell lysate. Lane 3: HA722780 IP in L-929 transfected with FLAG-tagged CD5 (C-terminal) cell lysate. Blocking/Dilution buffer: 5% NFDM/TBST Exposure time: 17 seconds; ECL: K1801
	Fig16: Immunohistochemical analysis of paraffin-embedded HeLa transfected with FLAG-tagged Histon H3.1 (N-terminal) cells with Rabbit anti-DYKDDDDK Tag (FLAG) antibody (HA722780) 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 (HA722780) 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.
	Fig17: Flow cytometric analysis of HeLa cells transfected with FLAG-tagged Histon H3.1 (N-terminal) labeling DYKDDDDK Tag (FLAG). Cells were fixed and permeabilized. Then stained with the primary antibody (HA722780, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

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