Anti-FLRT3 antibody [EPR29132-87]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-FLRT3 antibody [EPR29132-87] (AB323410)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized Huh7 (human hepatocellular carcinoma epithelial cell) cells labelling FLRT3 with ab323410 at 1/100 (5.04 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing cytoplasmic staining in Huh7 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Negative control : Jurkat.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/ 200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• WB

Supplier Data

Western blot - Anti-FLRT3 antibody [EPR29132-87] (AB323410)

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

Negative control : Jurkat.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 21673655, PMID : 24560577).

The identity of the bands around 50 kDa and 37KDa in lane 3 are unkown.

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

All lanes:

Western blot - Anti-FLRT3 antibody [EPR29132-87] (ab323410) at 1/1000 dilution

Lane 1:

A375 (human malignant melanoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

LN-229 (human brain glioblastoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Huh7 (human hepatocelullar carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) 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: 75-100 kDa,36 kDa

false

Exposure time: 114s

• WB

Supplier Data

Western blot - Anti-FLRT3 antibody [EPR29132-87] (AB323410)

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

FLRT3 is a glycoprotein of approximately 80-100 kDa and detected as a 75 kDa band after treated with Deglycosylation Peptide : N-glycosidase F (PNGase F).

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

All lanes:

Western blot - Anti-FLRT3 antibody [EPR29132-87] (ab323410) at 1/1000 dilution

Lane 1:

Untreated Huh7 (human hepatocellar carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Huh7 whole cell lysate treated with Protein Deglycosylation Peptide:N-glycosidase F (PNGase F) 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: 75-100 kDa,36 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-FLRT3 antibody [EPR29132-87] (AB323410)

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

Exposure time : Lanes 1-2 : 103 s; Lanes 3-4 : 148 s

This antibody was tested on fresh and frozen MEF and 4T1 whole cell lysate and it was concluded that FLRT3 is the easy target for degradation.

To minimize protein degradation, cells (lanes 3-4) were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

All lanes:

Western blot - Anti-FLRT3 antibody [EPR29132-87] (ab323410) at 1/1000 dilution

Lane 1:

MEF (mouse embryo fibroblast) whole cell lysate at 20 µg

Lane 2:

4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

MEF (mouse embryo fibroblast) fresh whole cell lysate at 20 µg

Lane 4:

4T1 (mouse mammary gland carcinoma epithelial cell) fresh 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: 75-100 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-FLRT3 antibody [EPR29132-87] (AB323410)

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

Low expression : heart, spleen(PMID : 10644439; PMID : 16872596)

The identity of the bands below 75 kDa are unkown.

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

All lanes:

Western blot - Anti-FLRT3 antibody [EPR29132-87] (ab323410) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 40 µg

Lane 2:

Mouse cerebellum tissue lysate at 40 µg

Lane 3:

Mouse hippocampus tissue lysate at 40 µg

Lane 4:

Mouse spleen tissue lysate at 40 µg

Lane 5:

Mouse heart tissue lysate at 40 µg

Lane 6:

Rat brain tissue lysate at 40 µg

Lane 7:

Rat P5 brain tissue lysate at 40 µg

Lane 8:

Rat spleen tissue lysate at 40 µg

Lane 9:

Rat heart tissue lysate at 40 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: 75-100 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-FLRT3 antibody [EPR29132-87] (AB323410)

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

This antibody does not cross-react with mouse FLRT1 and FLRT2.

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.

All lanes:

Western blot - Anti-FLRT3 antibody [EPR29132-87] (ab323410) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with an empty vector containing a His-tag, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a mouse FLRT3 expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a mouse FLRT1 expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 4:

293T cells transfected with a mouse FLRT2 expression vector containing a myc-His-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: 75-100 kDa,36 kDa

false

Exposure time: 1s