Recombinant Anti-GFAP antibody [RM1003] (ab278054)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized mouse primary neural/glia cells labelling GFAP with ab278054 at 1/500 (0.938 µg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green).
Confocal image showing cytoplasmic staining in mouse primary astrocytes. Confocal scanning Z step was set as 0.3 μm followed by image processing with maximum Z projection.

ab10062 Anti-GFAP mouse monoclonal antibody at 1/200 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) at a 1/500 dilution (Red) to counterstain. The nuclear counterstain was DAPI (Blue).

Negative control 1: ab278054 at a 1/500 dilution followed by ab150120 at a 1/500 dilution.

Negative control 2: ab10062 at a 1/500 dilution followed by ab150077 at a 1/1000 dilution.

Immunofluorescence staining of GFAP using ab278054 in primary rat hippocampal mixed glia, (prepared from P2 rat hippocampal brain area, obtained from Transnetyx Tissue by BrainBits, LLC, cat.no. SDPHP4m), DIV4. The cells were fixed with 100% MeOH (5 min), permeabilized with 0.1% Triton-X-100 (in PBS) for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab278054 at 0.1 μg/ml and ab4674, Anti-GFAP antibody, at 1/1000 dilution. Cells were then incubated with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150176, Goat Anti-Chicken IgY H&L (Alexa Fluor^® 594) preadsorbed at 1/1000 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown. The antibody ab278054 gave comparable results using 4% formaldehyde fixation (10 min).

All lanes : Anti-GFAP antibody [RM1003] (ab278054) at 1/1000 dilution

Lane 1 : U-87 MG (Human glioblastoma-astrocytoma epithelial cell) whole cell lysate
Lane 2 : SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate
Lane 3 : SK-N-BE (2) (Human neuroblastoma neuroblast) whole cell lysate
Lane 4 : IMR-32 (Human lung fibroblast) whole cell lysate
Lane 5 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 6 : Neuro-2a (Mouse neuroblastoma neuroblast) whole cell lysate
Lane 7 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate
Lane 8 : C6 (Rat glial tumor glial cell) whole cell lysate
Lane 9 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/20000 dilution

Predicted band size: 49 kDa
Observed band size: 40-54 kDa why is the actual band size different from the predicted?


Exposure time: 180 seconds

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

GFAP is expressed primarily by astrocytes in the CNS as astrocytes marker. We have no suitable astrocyte for western blot testing.
U-87 MG and C6 were reported to express low level of GFAP (PMID: 23839947, PMID: 22096544, PMID: 20669222).

NIH/3T3, IMR-32, HeLa and PC-12 were reported as negative cell lines for GFAP (PMID: 824020; PMID:2294; PMID: 6340792; PMID:9466565, PMID:7895062, PMID: 28700643, PMID: 19272755).

No literature was found to support the expression in SH-SY5Y, Neuro-2a and SK-N-BE (2) cell.

 

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized mouse cerebrum tissue labeling GFAP with ab278054 at 1/700 (0.938 µg/ml) dilution followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at a 1/1000 dilution. Nuclear counterstain is DAPI. Positive staining on mouse cerebrum. 

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) used at a 1/1000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling GFAP with ab278054 at 1/2000 (0.235 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human cerebrum. The section was incubated with ab278054 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 a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Flow Cytometry analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized mouse primary brain cells labeling GFAP using ab278054 at a 1/500 dilution (0.1 µg) (Right panel) compared to Rabbit monoclonal IgG (ab172730) (Left panel).

Secondary antibody is Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) used at a 1/2000 dilution.

All lanes : Anti-GFAP antibody [RM1003] (ab278054) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Rat brain lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 49 kDa
Observed band size: 40-54 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

The molecular weight observed is consistent with the literature (PMID: 25975510).

Blocking/Dilution buffer: 5% NFDM/TBST.

GFAP was immunoprecipitated from 0.35 mg mouse brain lysate with ab278054 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab278054 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Mouse brain lysate 10 μg.
Lane 2: ab278054 IP in mouse brain lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab278054 in mouse brain lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 seconds.
The molecular weight observed is consistent with the literature (PMID: 824020; PMID:2294; PMID: 6340792).

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling GFAP with ab278054 at 1/2000 (0.235 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse cerebrum. The section was incubated with ab278054 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 a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

All lanes : Anti-GFAP antibody [RM1003] (ab278054) at 1/1000 dilution

Lane 1 : Human brain lysate
Lane 2 : Human liver lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 49 kDa
Observed band size: 40-54 kDa why is the actual band size different from the predicted?


Exposure time: 3 seconds

Negative control: Human liver. (PMID: 25975510).
The molecular weight observed is consistent with the literature (PMID: 25975510).

Blocking/Diluting buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling GFAP with ab278054 at 1/2000 (0.235 µg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat cerebrum. The section was incubated with ab278054 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 a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.