Anti-Tau (O-GlcNAc S400) antibody [EPR24891-48]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Tau (O-GlcNAc S400) antibody [EPR24891-48] (AB320076)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T (human embryonic kidney epithelial cell) cells labelling Tau (O-GlcNAc S400) with ab320076 at 1/500 (1.02 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing positive staining in 293T cells transfected with a human Tau 2N4R expression vector containing a myc-His-tag®, and the signal increased after treatment with Thiamet G (10 um) for 48 h. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).
A : 293T (human embryonic kidney epithelial cell) cells transfected with a human wild-type Tau 2N4R expression vector containing a myc-His-tag®
B : 293T cells transfected with a human wild-type Tau 2N4R expression vector containing a myc-His-tag® were treated with 10 μM Thiamet G for 48 hours
C : 293T cells transfected with a human Tau 2N4R (S400A mutation) expression vector containing a myc-His-tag®
D : 293T cells transfected with a human Tau 2N4R (S400A mutation) expression vector containing a myc-His-tag® were treated with 10 μM Thiamet G for 48 hours

ab223894 Anti-Myc tag mouse monoclonal antibody (Alexa Fluor® 594) was used to counterstain tubulin at 1/100 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 2 ug/ml dilution.

• WB

Supplier Data

Western blot - Anti-Tau (O-GlcNAc S400) antibody [EPR24891-48] (AB320076)

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 22366723, PMID : 24113653).

The expression of Tau (O-GlcNAc S400) is upregulated in response to Thiamet G treatment (PMID : 25336656, PMID : 22366723, PMID : 24113653).

The identity of the higher MW band at approximately 110 kDa is unknown.

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

In Western blot, Anti-Tau antibody - Total protein control (ab76128) staining at 1/1000 dilution.

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

All lanes:

Western blot - Anti-Tau (O-GlcNAc S400) antibody [EPR24891-48] (ab320076) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with a human wild-type Tau 2N4R expression vector containing a myc-His-tag®, whole cell lysate at 50 µg

Lane 2:

293T cells transfected with a human wild-type Tau 2N4R expression vector containing a myc-His-tag® were treated with 10 uM Thiamet G for 48 hours, whole cell lysate at 50 µg

Lane 3:

293T cells transfected with a human Tau 2N4R (S400A mutation) expression vector containing a myc-His-tag®, whole cell lysate at 50 µg

Lane 4:

293T cells transfected with a human Tau 2N4R (S400A mutation) expression vector containing a myc-His-tag® were treated with 10 uM Thiamet G for 48 hours, whole cell lysate at 50 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: 25-75 kDa,36 kDa

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-Tau (O-GlcNAc S400) antibody [EPR24891-48] (AB320076)

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

The expression of Tau (O-GlcNAc S400) is reduced following deglycosylation induced by PNGase F.

The identity of the higher MW band at approximately 110 kDa is unknown.

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

All lanes:

Western blot - Anti-Tau (O-GlcNAc S400) antibody [EPR24891-48] (ab320076) at 1/1000 dilution

Lane 1:

293T cells transfected with a human wild-type Tau 2N4R expression vector containing a myc-His-tag® were treated with 10 uM Thiamet G for 48 hours 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® were treated with 10 uM Thiamet G for 48 hours whole cell lysate treated with 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: 25-75 kDa,36 kDa

false

Exposure time: 92s