Overview

  • Product name
    Anti-GFAP antibody [2A5]
    See all GFAP primary antibodies
  • Description
    Mouse monoclonal [2A5] to GFAP
  • Host species
    Mouse
  • Tested applications
    Suitable for: Sandwich ELISA, ICC, IHC-P, ICC/IF, WB, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human, Pig
    Predicted to work with: MammalsDoes not react with: Chicken
  • Immunogen

    Full length native protein (purified) corresponding to Pig GFAP. A preparation of purified pig spinal cord GFAP.

  • Positive control
    • Homogenized cow, pig, human, rat, mouse or other mammalian brain extract in 1%SDS, 6M urea.
  • General notes

    This clone gives a much stronger signal in pig, cow and human samples than in rodents. For rodent samples use ab68428.

Properties

Applications

Our Abpromise guarantee covers the use of ab4648 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Sandwich ELISA 1/5000.

Use this antibody as Capture antibody with Rabbit polyclonal to GFAP - Astrocyte Marker (ab48050) as Detection antibody at 0.5µg/ml.

ICC Use at an assay dependent concentration.
IHC-P 1/2000.
ICC/IF 1/10 - 1/50.
WB 1/100 - 1/500. Detects a band of approximately 55 kDa.
IHC-Fr Use at an assay dependent concentration.

Target

  • Function
    GFAP, a class-III intermediate filament, is a cell-specific marker that, during the development of the central nervous system, distinguishes astrocytes from other glial cells.
  • Tissue specificity
    Expressed in cells lacking fibronectin.
  • Involvement in disease
    Defects in GFAP are a cause of Alexander disease (ALEXD) [MIM:203450]. Alexander disease is a rare disorder of the central nervous system. It is a progressive leukoencephalopathy whose hallmark is the widespread accumulation of Rosenthal fibers which are cytoplasmic inclusions in astrocytes. The most common form affects infants and young children, and is characterized by progressive failure of central myelination, usually leading to death usually within the first decade. Infants with Alexander disease develop a leukoencephalopathy with macrocephaly, seizures, and psychomotor retardation. Patients with juvenile or adult forms typically experience ataxia, bulbar signs and spasticity, and a more slowly progressive course.
  • Sequence similarities
    Belongs to the intermediate filament family.
  • Post-translational
    modifications
    Phosphorylated by PKN1.
  • Cellular localization
    Cytoplasm. Associated with intermediate filaments.
  • Information by UniProt
  • Database links
  • Alternative names
    • wu:fb34h11 antibody
    • ALXDRD antibody
    • cb345 antibody
    • etID36982.3 antibody
    • FLJ42474 antibody
    • FLJ45472 antibody
    • GFAP antibody
    • GFAP_HUMAN antibody
    • gfapl antibody
    • Glial fibrillary acidic protein antibody
    • Intermediate filament protein antibody
    • wu:fk42c12 antibody
    • xx:af506734 antibody
    • zgc:110485 antibody
    see all

Images

  • ab4648 staining GFAP - Astrocyte Marker in pig brain tissue sections by IHC-Fr (formaldehyde-fixed Frozen sections). Tissue was fixed with formaldehyde (4%), permeabilized with Triton X-100 and blocked with 0.2% milk for 30 minutes at 24°C. Samples were incubated with primary antibody 1/2000 (TBS + 1% Triton X-100 + 0.2% milk) for 24 hours at 4°C. A Biotin-conjugated Sheep polyclonal to mouse IgG, dilution 1/400, was used as secondary antibody. Endogenous peroxidase was blocked with 0.3% H2O2 in methanol for 20 minutes.

    See Abreview

  • Rat neuron/glia cultures stained with nmouse monoclonal to GFAP ab 4648 (red).
  • Ab4648 staining human substantia nigra. Staining is localised to the cytoplasm.
    Left panel: with primary antibody diluted 1:4000. Right panel: isotype control.
    Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
  • Rat cortical neurons and glia in mixed tissue culture stained with Chicken polyclonal to MAP2 - ab5392 (green) at 1/30000 and Mouse monoclonal to GFAP - ab4648 (red) at 1/100. Nuclei of all cells are stained with Hoechst dye (blue). Picture taken with a Zeiss 20X objective and documented with a Digital SPOT camera.
  • Rat neuron/glia cultures stained with mouse monoclonal to GFAP ab4648 (red).
  • Standard Curve for GFAP; dilution range 1 pg/ml to 1 µg/ml using Capture Antibody Mouse monoclonal [2A5] to GFAP - Astrocyte Marker (ab4648) at 1/5000 and Detector Antibody Rabbit polyclonal to GFAP - Astrocyte Marker (ab48050) at 0.5 µg/ml.

  • Rat neuron/glia cultures stained with mouse monoclonal to GFAP ab4648 (green).

References

This product has been referenced in:
  • Rhodes CT  et al. Region specific knock-out reveals distinct roles of chromatin modifiers in adult neurogenic niches. Cell Cycle 17:377-389 (2018). Read more (PubMed: 29433384) »
  • Zhang T  et al. HMG-CoA Reductase Inhibitors Relieve Endoplasmic Reticulum Stress by Autophagy Inhibition in Rats With Permanent Brain Ischemia. Front Neurosci 12:405 (2018). Read more (PubMed: 29970982) »
See all 35 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (spinal cord)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 10 mM citrate, pH6.0
Permeabilization
Yes - 0.1% Tween 20 in PBS
Specification
spinal cord
Blocking step
Serum as blocking agent for 40 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Paraformaldehyde

Miss. Chia-Li Lin

Verified customer

Submitted Nov 24 2015

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: RTP°C
Sample
Mouse Cell (Mouse primary astrocyte)
Specification
Mouse primary astrocyte
Permeabilization
Yes - 0.1% Triton X-100
Fixative
Paraformaldehyde

Dr. Craig Beall

Verified customer

Submitted Sep 15 2014

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Brain)
Specification
Brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 26°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Nov 08 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Rat Cell (Embryonic rat cerebral cortex)
Specification
Embryonic rat cerebral cortex
Fixative
Paraformaldehyde
Permeabilization
Yes - Ice cold methanol
Blocking step
Serum as blocking agent for 15 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Dec 26 2012

Answer

Thank you for contacting Abcam.

Earlier we discussed your sELISA with ab4648 as the capture and ab48050 as the detector. As we discussed, since you would like to use your streptavidin-gold conjugated secondary, a detector that is conjugated to biotin is preferable so you can obtain the signal amplification necessary to have a detectable signal. Thus, we discussed testing ab79203 as your detector antibody through our AbTrial program. Your discount code and information about the abtrial program is given below:

I am very pleased to hear you would like to accept our offer and test ab79203 in sELISA. This code will give you $XXX off your next order before the expiration date. To redeem this offer, please submit an Abreview for sELISA and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active. Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: https://www.abcam.com/abtrial

On the phone I forgot to mention our current RabMab promotion, which entitles you to a free rabbit monoclonal antibody with any purchase of a primary antibody. Quote “RABMAB-XBSMG” in your next primary antibody order to take advantage of this offer. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Answer

The protocol used was as follows:
Buffers and Reagents:
Carbonate Coating Buffer (100 mM)
Antigen or antibody should be diluted in coating buffer to immobilize them to the wells:
3.03 g Na2CO3,
6.0 g NaHCO3
Make up to 1000 ml with distilled water and adjust to pH 9.6
PBS:
1.16 g Na2HPO4,
0.1 g KCl,
0.1 g K3PO4
4.0 g NaCl
Make up to 500 ml with distilled water and adjust to pH 7.4
Wash Solution - PBST (0.1% (v/v) Tween):
Add 5ml 10% (v/v) Tween-20 stock solution to 500ml PBS,
Blocking and Antibody/Protein Dilution Buffer:
5% (w/v) BSA in PBST.
Stop Solution:
0.25M Sulphuric Acid
Protocol:
1. 96-well ELISA plates are coated with 100µl/well of Capture Antibody ab4648 diluted 1:5000 in 100mM Carbonate Coating buffer, pH9.6.
2. Incubate the plates overnight at 4°C or 1 hour at room temperature to allow adsorption of the Capture Antibody to the plate.
3. Remove the Capture Antibody and was the plate 3 times with PBST (3x300µl/well), leaving the plate dry after the final wash.
4. Block the plates with 150µl/well of Blocking Buffer.
5. Incubate plates at room temperature for 1 hour.
6. Aspirate Blocking Buffer and add 50µl/well of Target Protein at the recommended concentration in Blocking Buffer.
7. Incubate for 1 hour at room temperature.
8. Remove the Protein Solution and was the plate 3 times with PBST (300µl/well), leaving the plate dry after the final wash.
9. Add 50µl/well of Detector Antibody ab48050 diluted to 0.5 ug/mL in Blocking Buffer.
10. Incubate for 1 hour at room temperature.
11. Remove the Detector Antibody solution and wash the plate 3 times with PBST (300µl/well), leaving the plate dry after the final wash.
12. Aliquot 100µl/well of anti-rabbit HRP conjugate Secondary Antibody diluted 1:10,000 in Blocking Buffer.
13. Incubate for 1 hour at room temperature.
14. Remove the Detector Antibody solution and was the plate 3 times with PBST (300µl/well), followed by 2 washes with PBS, leaving the plate dry after the final wash.
15. Add 50µl/well of TMB Peroxidase Substrate.
16. Incubate for 10 minutes at room temperature (The length of this incubation may be optimised, but 10 minutes works well in most cases).
17. Add 50µl/well of 0.25M Sulphuric Acid Stop Solution.
18. Read plates on plate reader at 450nm.

Read More

Question
Answer

We do recommend usingthe Sodium Citrate buffer (pH 6.0), however HIER are effective as well.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Pig Tissue sections (Brain)
Specification
Brain
Fixative
Formaldehyde
Permeabilization
Yes - Triton X-100
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 0.2% · Temperature: 24°C

Mr. Marian Hruska-Plochan

Verified customer

Submitted Feb 23 2010

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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