Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251]

• WB

Supplier Data

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking/Diluting buffer : 5% NFDM/TBST

Exposure time : 0.5 seconds

All lanes:

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1/1000 dilution

Lane 1:

HEK-293 (human epithelial cell line from embryonic kidney) transfected with an empty vector (vector control), containing an N-terminal DDDDK tag, whole cell lysate at 10 µg

Lane 2:

HEK-293 transfected with PFKFB3 (WT) expression vector containing an N-terminal DDDDK tag, whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 60 kDa

false

• WB

Supplier Data

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking and diluting buffer : 5% NFDM/TBST.

ab181602 was used as a loading control at 1/1000000 dilution.

ab205606 was used as a loading control at 1/1000 dilution.

This antibody shows very weak cross reactivity with ACSL5 and ACSL6.

All lanes:

Western blot - Anti-ACSL1 antibody [EPR13498] (<a href='/en-us/products/primary-antibodies/acsl1-antibody-epr13498-ab178419'>ab178419</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with an empty vector whole cell lysate at 10 µg

Lane 2:

293T transfected with a human ACSL1 expression vector containing a DDDDK tag whole cell lysate at 10 µg

Lane 3:

293T transfected with a human ACSL5 expression vector containing a DDDDK tag whole cell lysate

Lane 4:

293T transfected with a human ACSL6 expression vector containing a DDDDK tag whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 78 kDa

Observed band size: 75 kDa

false

Exposure time: 20s

• WB

Supplier Data

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking/Dilution buffer : 5% NFDM/TBST

Lane 1:

Western blot - Anti-ABCG1 antibody [EP1366Y] (<a href='/en-us/products/primary-antibodies/abcg1-antibody-ep1366y-ab52617'>ab52617</a>) at 1/1000 dilution

Lane 2:

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1/1000 dilution

All lanes:

C-Myc/DDK-tagged full length human ABCG1 recombinant protein at 10 ng

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking/Diluting buffer and concentration : 5% NFDM/TBST.

ab205606 can detect extra bands in non-transfected cell lines at low dilution.

All lanes:

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1.2 µg/mL

Lane 1:

HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 20 µg

Lane 3:

C6 (rat glial tumor cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/2000 dilution

Observed band size: 25 kDa,30 kDa,45 kDa,80 kDa

true

Exposure time: 20s

• WB

Supplier Data

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking buffer and concentration :  5% NFDM/TBST  Diluting buffer and concentration :  5% NFDM/TBST Exposure Time : Lane 1-2 : 1 second, Lane 3 : 40 seconds. ab181602 was used as loading controls. ab205606 was used as a total protein control. Tag constructed at C -terminal of overexpression vector may interfere the recognition of this antibody to targeted protein.

All lanes:

Western blot - Anti-KLHL6 antibody [EPR13484] (<a href='/en-us/products/primary-antibodies/klhl6-antibody-epr13484-ab182163'>ab182163</a>) at 1/1000 dilution

Lane 1:

293T (Human embryonic kidney epithelial cell) transfected with an empty vector (vector control) whole cell lysate at 20 µg

Lane 2:

293T transfected with KLHL6 expression vector containing a N-terminal DDDDK tag, whole cell lysate at 20 µg

Lane 3:

Human tonsil lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 70 kDa

Observed band size: 70 kDa

false

• WB

Lab

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

All lanes:

Western blot - Anti-ENO1 + ENO2 + ENO3 antibody [EPR3377] - Neuronal Marker (<a href='/en-us/products/primary-antibodies/eno1-eno2-eno3-antibody-epr3377-neuronal-marker-ab79757'>ab79757</a>) at 1/1000 dilution

Lane 1:

Recombinant human ENO1 protein with C-DDDDK tag at 10 ng

Lane 2:

Recombinant human ENO2 protein with C-DDDDK tag at 10 ng

Lane 3:

Recombinant human ENO3 protein with C-DDDDK tag at 10 ng

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 50 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

All lanes:

Western blot - Anti-NSE antibody [EPR12483] - Neuronal Marker (<a href='/en-us/products/primary-antibodies/nse-antibody-epr12483-neuronal-marker-ab180943'>ab180943</a>) at 1/10000 dilution

Lane 1:

Recombinant human ENO1 protein with C-DDDDK tag at 10 ng

Lane 2:

Recombinant human ENO2 (NSE) protein with C-DDDDK tag at 10 ng

Lane 3:

Recombinant human ENO3 protein with C-DDDDK tag at 10 ng

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 50 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Lane 1:

GST-tagged human CLIP3/CLIPR-59 recombinant full-length protein at 10 ng

Lane 2:

GST-tagged human CLIP4 recombinant full-length protein at 10 ng

Lane 3:

GST-tagged human CLIP2 recombinant full-length protein at 10 ng

Lane 4:

DDDDK-tagged human CLIP1/CLIP170 HEK293T cell transient overexpression lysate at 10 ng

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 86 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking and dilution buffer : 5% NFDM/TBST.

Anti-DDDDK tag (Binds to FLAG^® tag sequence) antibody (ab205606) used as loading control at 1/1000 dilution. The secondary antibody used is ab97051, a HRP goat anti-rabbit IgG (H+L), at a dilution of 1/20000.

All lanes:

Western blot - Anti-eIF4A3 antibody [EPR14301(B)] (<a href='/en-us/products/primary-antibodies/eif4a3-antibody-epr14301b-ab180573'>ab180573</a>) at 1/1000 dilution

Lane 1:

eIF4A1 Human Recombinant Protein at 10 ng

Lane 2:

eIF4A2 Human Recombinant Protein at 10 ng

Lane 3:

eIF4A3 Human Recombinant Protein at 10 ng

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 47 kDa

Observed band size: 46 kDa

false

Exposure time: 5s

• WB

Supplier Data

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking and diluting buffer concentration : 5% NFDM/TBST.

ab205606 DDDDK tag was used as a loading control at 1/1000 dilution.

All lanes:

Western blot - Anti-IMP3 antibody [EPR12021-114] (<a href='/en-us/products/primary-antibodies/imp3-antibody-epr12021-114-ab179807'>ab179807</a>) at 1/1000 dilution

Lane 1:

C-Myc/DDK -tagged full length Human IGF2BP1 recombinant protein at 0.01 µg

Lane 2:

C-Myc/DDK -tagged full length Human IGF2BP2 recombinant protein at 0.01 µg

Lane 3:

N-His -tagged full length Human IGF2BP3 recombinant protein at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 63 kDa

Observed band size: 75 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking and diluting buffer : 5% NFDM/TBST.

ab205606 was used as a loading control at 1/1000 dilution.

ab179807 was used at 1/1000 dilution.

Lanes 1 - 3:

Western blot - Anti-IGF2BP2/IMP-2 antibody [EPR6741(B)] (<a href='/en-us/products/primary-antibodies/igf2bp2-imp-2-antibody-epr6741b-ab124930'>ab124930</a>) at 1/1000 dilution

Lanes 1 - 3:

Western blot - Anti-IMP3 antibody [EPR12021-114] (<a href='/en-us/products/primary-antibodies/imp3-antibody-epr12021-114-ab179807'>ab179807</a>) at 1/1000 dilution

Lane 1:

C-Myc/DDK-tagged full length Human IGF2BP1 recombinant protein at 0.01 µg

Lane 2:

C-Myc/DDK-tagged full length Human IGF2BP2 recombinant protein at 0.01 µg

Lane 3:

N-His-tagged full length Human IGF2BP3 recombinant protein at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 66 kDa

Observed band size: 75 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking and diluting buffer : 5% NFDM/TBST.

This antibody shows weak cross reactivity with IGF2BP2.

ab205606 was used as a loading control at 1/1000 dilution.

ab179807 was used at 1/1000 dilution.

Lanes 1 - 3:

Western blot - Anti-IGF2BP1/IMP1 antibody [EPR18791] (<a href='/en-us/products/primary-antibodies/igf2bp1-imp1-antibody-epr18791-ab184305'>ab184305</a>) at 1/1000 dilution

Lanes 1 - 3:

Western blot - Anti-IMP3 antibody [EPR12021-114] (<a href='/en-us/products/primary-antibodies/imp3-antibody-epr12021-114-ab179807'>ab179807</a>) at 1/1000 dilution

Lane 1:

C-Myc/DDK-tagged full length Human IGF2BP1 recombinant at 0.01 µg

Lane 2:

C-Myc/DDK-tagged full length Human IGF2BP2 recombinant protein at 0.01 µg

Lane 3:

N-His-tagged full length Human IGF2BP3 recombinant protein at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 63 kDa

Observed band size: 75 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

All lanes:

Western blot - Anti-PCBP2/hnRNP E2 antibody [EPR14859(2)] (<a href='/en-us/products/primary-antibodies/pcbp2-hnrnp-e2-antibody-epr148592-ab200835'>ab200835</a>) at 1/1000 dilution

All lanes:

Recombinant Mouse Pcbp2 Protein, with C-term MYC/DDK tag at 10 ng

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 50 kDa

false

Exposure time: 120s

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) cells labeling DDDDK tag with ab205606 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (green).

Confocal image showing positive staining for FLAG® on HEK-293T cells transfected with DDDDK-tagged PFKFB3 expression vector. Mouse monoclonal ANTI-FLAG^® M2 antibody was used as a counterstain at 1/500 dilution, and AlexaFluor^®647 Goat anti-mouse secondary (ab150115) was used as the secondary antibody only control at 1/500 dilution. The nucleus is counterstained with DAPI.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Immunhistochemical analysis of agarose-embedded HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with DDDDK-tagged human PD-L1 expression vector labeling DDDDK tag with ab205606 at 1/750 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Counterstained with hematoxylin.

Positive staining on HEK-293T cells transfected with DDDDK-tagged human PD-L1 expression vector (Panel A) is observed. No signal was detected on HEK-293T transfected with an empty vector (vector control), containing a C-terminal DDDDK tag (Panel B).

Heat mediated antigen retrieval was performed using EDTA buffer pH 9 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Negative control : No staining on mouse spleen.

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue stained for DDDDK tag using ab205606 at 1/750 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Counterstained with hematoxylin.

Heat mediated antigen retrieval was performed using EDTA buffer pH 9 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Negative control : No staining on rat spleen.

Immunohistochemical analysis of paraffin-embedded rat spleen tissue stained for DDDDK tag using ab205606 at 1/750 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Counterstained with hematoxylin.

Heat mediated antigen retrieval was performed using EDTA buffer pH 9 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Negative control : No staining on human hepatocellular cancer.

Immunohistochemical analysis of paraffin-embedded human hepatocellular cancer tissue stained for DDDDK tag using ab205606 at 1/750 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Counterstained with hematoxylin.

Heat mediated antigen retrieval was performed using EDTA buffer pH 9 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

DDDDK tag was immunoprecipitated from 0.35 mg HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) transfected with DDDDK-tagged human PFKFB3 expression vector whole cell lysate with ab205606 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab205606 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HEK-293T transfected with DDDDK-tagged human PFKFB3 expression vector whole cell lysate (input).

Lane 2 : ab205606 IP in HEK-293T transfected with DDDDK-tagged human PFKFB3 expression vector whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab205606 in HEK-293T transfected with DDDDK-tagged human PFKFB3 expression vector whole cell lysate.

Blocking/Dilution buffer : 5% NFDM/TBST
Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606)

false

• WB

Lab

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking and dilution buffer and concentration : 5% NFDM/TBST. ab205606 was also used at 1/1000 dilution to detect C-Myc/DDK tagged human S6K1 and S6K2 recombinant proteins shown on the right panel. ab32539 showed cross reactivity with S6K2 in recombinant protein testing. S6K1 and S6K2 have different observed band size in endogenous and recombinant protein tests.

Lanes 1 - 2:

Western blot - Anti-S6K1 antibody [E175] (<a href='/en-us/products/primary-antibodies/s6k1-antibody-e175-ab32359'>ab32359</a>) at 1/1000 dilution

Lanes 1 - 2:

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1/1000 dilution

Lane 1:

C-Myc/DDK tagged human S6K1 recombinant protein at 10 ng

Lane 2:

C-Myc/DDK tagged human S6K2 recombinant protein at 10 ng

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 59 kDa

Observed band size: 75 kDa,60 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking and diluting buffer and concentration : 5% NFDM /TBST.

ab205606 was used as a loading control.

All lanes:

Western blot - Anti-iNOS+nNOS antibody [EP16634] (<a href='/en-us/products/primary-antibodies/inosnnos-antibody-ep16634-ab202417'>ab202417</a>) at 1/1000 dilution

Lane 1:

C-Myc/DDK tagged full length human NOS1 (nNOS) recombinant protein at 10 ng

Lane 2:

C-Myc/DDK tagged full length human NOS2 (iNOS) recombinant protein at 10 ng

Lane 3:

C-Myc/DDK tagged full length human NOS3 (eNOS) recombinant protein at 10 ng

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 130 kDa,160 kDa

false

Exposure time: 20s

• WB

Supplier Data

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This antibody does not cross-react with human MAP2 and MAP4.

The antibody binds in the region surrounding Leu243 of 2N4R Tau

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

In Western blot, Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody(ab205606) staining at 37-250KDa dilution.

All lanes:

Western blot - Anti-Tau (MTBR-tau243) antibody [EPR29541-59] (<a href='/en-us/products/primary-antibodies/tau-mtbr-tau243-antibody-epr29541-59-ab322554'>ab322554</a>) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human wild-type Tau 2N4R expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a human wild-type PNS-Tau expression vector containing a DDDDK-tag, whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a human wild-type MAP2 expression vector containing a DDDDK-tag, whole cell lysate at 20 µg

Lane 5:

293T cells transfected with a human wild-type MAP4 expression vector containing a DDDDK-tag, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 33-100 kDa,36 kDa,15-150 kDa,37-250 kDa

false

Exposure time: 6s

• WB

Supplier Data

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking/Diluting buffer : 5% NFDM/TBST

Exposure time : 1 second

All lanes:

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1/5000 dilution

Lane 1:

HEK-293 (human epithelial cell line from embryonic kidney) transfected with an empty vector (vector control), containing a C-terminal DDDDK tag, whole cell lysate at 10 µg

Lane 2:

HEK-293 transfected with PD-L1 (WT) expression vector containing a C- terminal DDDDK tag, whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

false

• WB

Lab

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking and diluting buffer and concentration : 5% NFDM /TBST

ab181602 was used as a GAPDH loading control at 1/1000000 dilution.

ab205606 was used at 1/1000 dilution.

All lanes:

Western blot - Anti-ACSL1 + ACSL6 antibody [EPR13499] (<a href='/en-us/products/primary-antibodies/acsl1-acsl6-antibody-epr13499-ab177958'>ab177958</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with an empty vector whole cell lysate at 10 µg

Lane 2:

293T transfected with a human ACSL1 expression vector containing a DDDDK tag whole cell lysate at 10 µg

Lane 3:

293T transfected with a human ACSL5 expression vector containing a DDDDK tag whole cell lysate at 10 µg

Lane 4:

293T transfected with a human ACSL6 expression vector containing a DDDDK tag whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/200000 dilution

Observed band size: 75 kDa

false

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking and diluting buffer and concentration : 5% NFDM /TBST. Exposure time : Lane 1, 2, 4, 5 , 6 : 1 second. Lane 3 : 80 seconds.

Lanes 1 - 3:

Western blot - Anti-IRS1+IRS2 antibody [EP263Y] (<a href='/en-us/products/primary-antibodies/irs1irs2-antibody-ep263y-ab40777'>ab40777</a>) at 1/1000 dilution

Lanes 4 - 6:

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) at 1/1000 dilution

Lanes 1 and 4:

HEK-293T transfected with an empty vector whole cell lysate at 20 µg

Lanes 2 and 5:

HEK-293T transfected with a human IRS1 expression vector containing a Myc/DDK tag whole cell lysate at 20 µg

Lanes 3 and 6:

HEK-293T transfected with a human IRS2 expression vector containing a Myc/DDK tag whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 170 kDa

false

• WB

Supplier Data

Western blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

This antibody does not cross-react with human MAP2 and MAP4.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-Tau (N-terminal) antibody [EPR29521-8] (<a href='/en-us/products/primary-antibodies/tau-n-terminal-antibody-epr29521-8-ab324236'>ab324236</a>) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human Tau 2N4R expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a human PNS-Tau expression vector containing a DDDDK-tag, whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a human MAP2 expression vector containing a DDDDK-tag, whole cell lysate at 20 µg

Lane 5:

293T cells transfected with a human MAP4 expression vector containing a DDDDK-tag, whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/100000 dilution

Observed band size: 33-60 kDa,15-150 kDa,37-250 kDa,36 kDa

false

Exposure time: 1s

• Dot

Supplier Data

Dot Blot - Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (AB205606)

Dot blot analysis of pan NOS using ab316334 at 1 : 1000 (0.503 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 20,000 dilution.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Anti-DDDDK tag (Binds to FLAG® tag sequence) antibody [EPR20018-251] (ab205606) as total protein control staining at 1/1000 dilution.

All lanes:

Dot Blot - Anti-pan NOS antibody [RM1126] (<a href='/en-us/products/primary-antibodies/pan-nos-antibody-rm1126-ab316334'>ab316334</a>) at 1/1000 dilution

Lane 1:

C-Myc/DDK tagged full length human nNOS recombinant protein

Lane 2:

C-Myc/DDK tagged full length human iNOS recombinant protein

Lane 3:

C-Myc/DDK tagged full length human eNOS recombinant protein

Secondary

All lanes:

Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

false

Exposure time: 1s