Anti-TIE2 antibody [EPR21915]

• Flow Cyt

Unknown

Flow Cytometry - Anti-TIE2 antibody [EPR21915] (AB221154)

Flow cytometric analysis of HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) (left panel) and HUVEC (human umbilical vein endothelial cell line) (right panel) cells labeling TIE2 with ab221154 at 1/500 dilution (red) compared with a Rabbit monoclonal IgG (ab172730) Isotype control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.

Negative control : HEK - 293T (PMID : 17189382)

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-TIE2 antibody [EPR21915] (AB221154)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HUVEC (human umbilical vein endothelial cell line) cells labeling TIE2 with ab221154 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous and cytoplasmic staining in HUVEC cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Control : Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Negative control : HEK-293T (PMID : 17189382).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-TIE2 antibody [EPR21915] (AB221154)

Flow cytometry overlay histogram showing positive HUVEC cells (left) and negative Hek-293 cells (right) stained with ab221154 (red line). The cells were fixed with 4% PFA and permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab221154) (1x 10⁶ in 100μl at 0.2 μg/ml (1/2,500)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C.

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (black line). Unlabelled sample was also used as a control (blue line).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• I-ELISA

Lab

Indirect ELISA - Anti-TIE2 antibody [EPR21915] (AB221154)

Indirect ELISA showing primary antibody ab221154 binding to mouse TEK and Human TEK. Antigen concentration is 1000 ng/ml. Binding of ab221154 was assessed in a serial dilution range 1000-0 ng/ml. Binding was detected using the secondary antibody, Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution.

• WB

Supplier Data

Western blot - Anti-TIE2 antibody [EPR21915] (AB221154)

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

The identity of the bands lower than 100 kDa are unknown.

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

In Western blot, Anti-TIE2 antibody [EPR21915] - (ab221154) staining at 1/500 dilution.

All lanes:

Western blot - Anti-TIE2 (phospho Y992) antibody [EPR27958-1] (<a href='/en-us/products/primary-antibodies/tie2-phospho-y992-antibody-epr27958-1-ab321883'>ab321883</a>) at 1/1000 dilution

Lane 1:

Untreated HUVEC (human umbilical vein endothelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

HUVEC treated with 100 uM Pervanadate for 10 minutes whole cell lysate (untreated membrane) at 20 µg

Lane 3:

HUVEC treated with 100 uM Pervanadate for 10 minutes whole cell lysate (alkaline phosphatase treated membrane) 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: 36 kDa,126 kDa

false

Exposure time: 103s

• WB

Lab

Western blot - Anti-TIE2 antibody [EPR21915] (AB221154)

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

To preserve protein degradation, harvested cells were immediately lysed, followed by rapid gel electrophoresis and membrane transfer procedures.

All lanes:

Western blot - Anti-TIE2 antibody [EPR21915] (ab221154) at 1/1000 dilution

Lane 1:

HUVEC (human umbilical vein endothelial cell line) fresh whole cell lysate at 20 µg

Lane 2:

HUVEC (human umbilical vein endothelial cell line) frozen whole cell lysate at 20 µg

Lane 3:

Mouse lung fresh tissue lysate at 20 µg

Lane 4:

Mouse lung frozen tissue 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: 160 kDa,60 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-TIE2 antibody [EPR21915] (AB221154)

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

The antibody ab221154 exhibits low sensitivity in Western blot analyses. To improve detection of full-length TIE2, we recommend substituting with ab307556 or ab259982. Alternatively, experimental conditions can be optimized through increasing sample loading quantity, using lower antibody dilution or implementing femtogram-level sensitivity detection substrates.

Important note : TIE2 undergoes proteolytic cleavage (PMID : 32838837, PMID : 26666854). These three antibodies target distinct cleaved fragments of TIE2 due to differences in their immunogen localization.

Lane 1:

Western blot - Anti-TIE2 antibody [EPR21915] (ab221154)

Lane 2:

Western blot - Anti-TIE2 antibody [EPR26540-78] (<a href='/en-us/products/primary-antibodies/tie2-antibody-epr26540-78-ab307556'>ab307556</a>)

Lane 3:

Western blot - Anti-TIE2 antibody [EPR23823-109] (<a href='/en-us/products/primary-antibodies/tie2-antibody-epr23823-109-ab259982'>ab259982</a>)

All lanes:

HUVEC (human umbilical vein endothelial 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 1/20000 dilution

Observed band size: 60 kDa,40 kDa,160 kDa

false

Exposure time: 40s

• WB

Unknown

Western blot - Anti-TIE2 antibody [EPR21915] (AB221154)

Blocking and dilution buffer : 5% NFDM/TBST.

The molecular mass observed is consistent with what has been described in the literature (PMID : 26666854). The antibody detects multiple bands (≤ 50 kDa) which are likely to be cleavage fragments of Tie2.

Negative control : HEK-293T (PMID : 15851516).

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-TIE2 antibody [EPR21915] (ab221154) at 1/100000 dilution

Lane 1:

Human lung tissue lysate at 20 µg

Lane 2:

HUVEC (human umbilical vein endothelial cell line) whole cell lysate at 20 µg

Lane 3:

U-87 MG (human glioblastoma-astrocytoma epithelial cell line) whole cell lysate at 20 µg

Lane 4:

HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 125 kDa

Observed band size: 40 kDa,50 kDa,160 kDa

true

Exposure time: 3min

• WB

Lab

Western blot - Anti-TIE2 antibody [EPR21915] (AB221154)

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

The antibody ab221154 exhibits low sensitivity in Western blot analyses. To improve detection of full-length TIE2 in mouse samples, we recommend substituting with ab307556. Alternatively, experimental conditions can be optimized through increasing sample loading quantity, using lower antibody dilution or implementing femtogram-level sensitivity detection substrates.

To preserve protein degradation, harvested cells were immediately lysed, followed by rapid gel electrophoresis and membrane transfer procedures.

Lanes 1 and 3:

Western blot - Anti-TIE2 antibody [EPR21915] (ab221154) at 1/1000 dilution

Lanes 2 and 4:

Western blot - Anti-TIE2 antibody [EPR26540-78] (<a href='/en-us/products/primary-antibodies/tie2-antibody-epr26540-78-ab307556'>ab307556</a>) at 1/1000 dilution

Lanes 1 - 2:

bEnd.3 (mouse brain endothelial cell) fresh whole cell lysate at 20 µg

Lanes 3 - 4:

bEnd.3 (mouse brain endothelial cell) frozen 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: 160 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-TIE2 antibody [EPR21915] (AB221154)

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

The antibody ab221154 exhibits low sensitivity in Western blot analyses. To improve detection of full-length TIE2 in mouse samples, we recommend substituting with ab307556. Alternatively, experimental conditions can be optimized through increasing sample loading quantity, using lower antibody dilution or implementing femtogram-level sensitivity detection substrates.

Lane 1:

Western blot - Anti-TIE2 antibody [EPR21915] (ab221154) at 1/1000 dilution

Lane 2:

Western blot - Anti-TIE2 antibody [EPR26540-78] (<a href='/en-us/products/primary-antibodies/tie2-antibody-epr26540-78-ab307556'>ab307556</a>) at 1/1000 dilution

All lanes:

Mouse lung tissue 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: 160 kDa

false

Exposure time: 180s