Anti-GFAP (phospho S13) antibody [EPR25281-65]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFAP (phospho S13) antibody [EPR25281-65] (AB303560)

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling GFAP (phospho S13) with ab303560 at 1/500 dilution (1.016 µg/mL), followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining was observed on astrocyte in human cerebrum without alkaline phosphatase treatment (image A, PMID : 22528834). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab303560 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) at Ready to use dilution. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-GFAP (phospho S13) antibody [EPR25281-65] (AB303560)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized U-87 MG (human glioblastoma-astrocytoma epithelial cell) treated with 1ug/ml LPS for 24 hours, then add 100nM Calyculin A for 30 min (Red) / Untreated control (Green) cells labelling GFAP (phospho S13) with ab303560 at 1/500 dilution (0.1ug) (Red) and Green (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• WB

Supplier Data

Western blot - Anti-GFAP (phospho S13) antibody [EPR25281-65] (AB303560)

Blocking and dilution buffer and concentration : 5% NFDM/TBST.  The molecular weight observed is consistent with what has been described in the literature (PMID : 31682229, PMID : 22528834).

All lanes:

Western blot - Anti-GFAP (phospho S13) antibody [EPR25281-65] (ab303560) at 1/1000 dilution

Lane 1:

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

Lane 2:

U-87 MG treated with 1ug/ml LPS for 24 hours, then add 100nM Calyculin A for 30 min, 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

Predicted band size: 49 kDa

Observed band size: 49 kDa

false

Exposure time: 70s

• WB

Supplier Data

Western blot - Anti-GFAP (phospho S13) antibody [EPR25281-65] (AB303560)

Blocking and dilution buffer and concentration : 5% NFDM/TBST.  The molecular weight observed is consistent with what has been described in the literature (PMID : 31682229, PMID : 22528834).

All lanes:

Western blot - Anti-GFAP (phospho S13) antibody [EPR25281-65] (ab303560) at 1/1000 dilution

Lane 1:

U-87 MG (human glioblastoma-astrocytoma epithelial cell) treated with 1 ug/ml LPS for 24 hours, then add 100 nM Calyculin A for 30 min, whole cell lysate (untreated membrane) at 20 µg

Lane 2:

U-87 MG treated with 1 µg/ml LPS for 24h, then add 100 nM Calyculin A for 30 min, (phosphatase treated membrane), 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

Predicted band size: 49 kDa

Observed band size: 35 kDa,55 kDa,50 kDa

false

Exposure time: 70s

• WB

Supplier Data

Western blot - Anti-GFAP (phospho S13) antibody [EPR25281-65] (AB303560)

Blocking and dilution buffer and concentration : 5% NFDM/TBST.  Exposure time : 5.5 seconds.

All lanes:

Western blot - Anti-GFAP (phospho S13) antibody [EPR25281-65] (ab303560) at 1/1000 dilution

Lane 1:

HEK-293T (human embryonic kidney cell) transfected with a human GFAP (WT) expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 2:

HEK-293T transfected with a human GFAP (S13A mutation) 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 1/100000 dilution

Predicted band size: 49 kDa

Observed band size: 35 kDa,55 kDa,50 kDa

false

Exposure time: 5.5s

• Dot

Supplier Data

Dot Blot - Anti-GFAP (phospho S13) antibody [EPR25281-65] (AB303560)

Dot blot analysis of GFAP (phospho S13) using ab303560 at 1 : 1000 (0.508 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution. Exposure time : 180 seconds Blocking and diluting buffer and concentration : 5% NFDM/TBST