Anti-GFAP antibody ValidAbTM

(HB7592)
Technical documents: SDS Datasheet

Product overview

Name Anti-GFAP antibody ValidAbTM
Host Goat
Clonality Polyclonal
Target GFAP
Description

Antibody to GFAP - cytoskeletal protein used as an astrocyte marker. Part of the ValidAb™ range of highly validated, data-rich antibodies.

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Validation data

Figure 1. Astrocytes in culture in a cultured rat neuron preparation.

HB7592 stains GFAP in the star shaped projections of an astrocyte. Method: neurons were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilized with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7592 was incubated overnight (4°C) at a 1:2,000 dilution (0.5µg/ml). This was followed by a one hour incubation with secondary antibody (Polyclonal donkey anti-goat DyLight 594 conjugated, Thermofisher SA5-10088, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualize cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope. The image was captured as a tilescan in Lightning deconvolution mode using a 63x objective (4.1x zoom), 405nm (21.1% power, PMT: 512V gain) and 561nm (0.1% power, Hyd: 10% gain) laser lines in a z-stack (0.39µm spacing). The stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 2. Astrocyte populations in culture stained by HB7592.

HB7592 stains the populations of glia found in a cultured rat neuron preparation. Method: neurones were cultured from E17-E18 rat embryos following established protocols (Martin and Henley, 2004. EMBO, 4749–4759) and fixed with 4% PFA on DIV21. Cells were permeabilized with 0.1% Triton X-100 followed by blocking in 1% BSA, 300mM glycine. HB7592 was incubated overnight (4°C) at a 1:2,000 dilution (0.5µg/ml). This was followed by a one hour incubation with secondary antibody (Polyclonal donkey anti-goat DyLight 594 conjugated, Thermofisher SA5-10088, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualize cell nuclei. For more detail please see our ICC protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured using a 10x objective (DAPI: 144.1ms, RHO, 57.4ms exposures) in a z-stack (4.3µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 3. Astrocytes stained for GFAP with HB7592 in the cerebellum

Rat cerebellum stained by HB7592 for GFAP. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before HB7592 was incubated overnight (4°C) at a 1:2,000 dilution (0.5µg/ml). This was followed by a two hour incubation with secondary antibody (Polyclonal donkey anti-goat DyLight 594 conjugated, Thermofisher SA5-10088, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 20x objective (DAPI: 106.6ms, Y3: 349.1ms exposures) in a z-stack (2.4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 4. Independent antibody validation of HB7592 and HB6406.

The pattern of staining for HB7592 and HB6406 in the cerebellum overlaps giving strong evidence for specificity. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before HB7592 was incubated overnight (4°C) at a 1:2,000 dilution (0.5µg/ml) with HB6406 also at a 1:2,000 dilution. This was followed by a two hour incubation with secondary antibodies (Polyclonal donkey anti-goat DyLight 594 conjugated, Thermofisher SA5-10088, 1:300 dilution and polyclonal donkey anti-chicken Alexa Fluor 488, Thermofisher A78948, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualise cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera. The image was captured as a tilescan using a 20x objective (DAPI: 106.6ms, GFP: 83.7ms, Y3: 349.1ms exposures) in a z-stack (2.4µm spacing). The image was deconvolved in Huygens professional software before the stack was flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Figure 5. Concentration response of HB7592 staining in cultured rat neurons.

HB7592 produces strong staining of astrocytes in rat cerebellum sections at dilutions down to 1:8,000. Method: Rat brains were dissected and fixed overnight in 4% PFA before then being incubated in 30% sucrose (in PBS) until sunk (approx. 48hrs). A freezing microtome was used to cut 40µm horizontal slices before sections were incubated in 1% NaBH4 for 30 minutes. Sections were blocked in 0.05M glycine, 2% BSA and 3% donkey serum before incubation overnight in HB752 dilutions ranging from 1:2,000 to 1:16,000 (0.0625 – 0.5µg/ml). This was followed by a two hour incubation with a polyclonal donkey anti-goat DyLight 594 conjugated secondary antibody (Thermofisher SA5-10088, 1:300 dilution). DAPI (HB0747) was used at 1µg/ml to visualize cell nuclei. For more detail please see our IHC(IF) protocol. Images were captured using a Leica DMI6000B inverted epifluorescence microscope connected to a Photometric Prime 95B camera as a z-stack with a 40x objective before being deconvolved in Huygens professional software and flattened using a maximum Z projection in ImageJ (Schindelin et al., 2012. Nat Methods, 9(7), 676–682).

Exposure times were as follows:

  • 1:2,000: DAPI: 10.0ms, TX2: 38.4ms
  • 1:4,000: DAPI: 10.0ms, TX2: 44.1ms
  • 1:8,000: DAPI: 10.0ms, TX2: 44.1ms
  • 1:16,000: DAPI: 10.0ms, TX2: 94.5ms


Product information

Immunogen

Recombinant human GFAP (isoform 1) expressed in and purified from E. coli

Purification

Immunogen affinity purification

Concentration 1mg/ml
Formulation 50% PBS, 50% glycerol + 5mM sodium azide
Predicted species reactivity Mouse, Rat, Human
Tested species reactivity Mouse, Rat

Tested applications

Applications ICC, IHC(IF)
IHC(IF) optimal concentration

0.5µg/ml (1:2,000 dilution) as tested in free-floating paraformaldehyde fixed rat brain sections

ICC optimal concentration

0.5µg/ml (1:2,000 dilution) as tested in cultured rat neurones

Positive control

GFAP is highly expressed in neural tissues containing astrocytes. It is not widely expressed in cell lines, however it is in specific lines such as U-87 MG.

Negative control

Most non-neural tissues.
Please note that GFAP expression has been reported in a subset of pancreatic and hepatic cells in rats and mice kidney cells. It is generally poorly expressed in common cell lines such as HeLa or HEK293.

Open data link

Please follow this link to OSF

Target information

UniProt ID P14136
Structure image  Chemical Structure
Gene name GFAP
NCBI full gene name glial fibrillary acidic protein
Entrez gene ID

2670

Amino acids

432 (49.9kDa)

Isoforms

GFAP has three confirmed and 21 potential isoforms. Isoform 1 (GFAP alpha): canonical, 49.9kDa; Isoform 2 (GFAP epsilon): amino acid changes between positions 391 and 432, 49.5kDa; Isoform 3 (GFAP kappa): amino acid changes between positions 391 and 432, 50.3kDa

Expression

GFAP is primarily expressed within astrocytes of the central nervous system alongside also expressing in non-myelinating Schwann cells of the peripheral nervous system and satellite cells of the peripheral ganglia. GFAP expression has also been reported in Leydig cells of the testis alongside stellate cells from the pancreas and liver in rats.

Subcellular expression

GFAP is a key cytoskeletal component therefore is widely expressed as bundles of GFAP positive fibres.

Processing

Following translation, no processing is required for GFAP to reach its active conformation.

Post translational modifications

GFAP is subjected to numerous post-translational modifications including 9 phosphorylation sites which are the target of AURKB and ROCK1 alongside 5 separate citrullination sites.

Homology (compared to human)

Rat, mouse and human GFAP proteins have a 90% similarity score in a direct BLAST comparison.

Similar proteins

Other type III intermediate filament proteins have homology with GFAP including Vimentin (58%), Desmin (59%) and Peripherin (56%) when assessed using BLAST.

Storage & Handling

Storage instructions

-20°C

Important This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use

FAQs

What mounting media do you recommend to use with this antibody?
What guarantee do you have that my GFAP antibody will perform as expected?

We guarantee that your GFAP antibody will work for the applications and species we list on the datasheet. If the antibody fails to perform as expected then we are happy to offer a 100% refund guarantee. For more details please see our guarantee policy.

Will my GFAP antibody work against species that have not been listed on the datasheet?

A species not being listed doesn’t mean that the GFAP antibody won’t work, just that we haven’t tested it. If you test one of our antibodies in a new species please let us know (positive or negative)!

What protocols are available for use with this GFAP antibody

We have made a comprehensive collection of protocols that we have used in our experiments to validate this GFAP antibody.

Any other questions?

For any other questions about our antibody products please see our technical FAQs for antibodies

References for Anti-GFAP antibody ValidAbTM

References are publications that support the biological activity of the product
  • Importance of GFAP isoform-specific analyses in astrocytoma.

    van Bodegraven EJ et al (2019) Glia 67 : 1417-1433
  • The role of GFAP and vimentin in learning and memory.

    Wilhelmsson U et al (2019) Biological chemistry 400 : 1147-1156
  • GFAP-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain.

    Garcia AD et al (2004) Nature neuroscience 7 : 1233-41
  • Glial fibrillary acidic protein: GFAP-thirty-one years (1969-2000).

    Eng LF et al (2000) Neurochemical research 25 : 1439-51
  • GFAP and astrogliosis.

    Eng LF et al (1994) Brain pathology (Zurich, Switzerland) 4 : 229-37