Figure 1: GFAP protein structure.

GFAP Introduction

Protein Function

• GFAP is the hallmark intermediate filament protein in astrocytes, the main type of glial cells in the central nervous system. It serves as a cell-type-specific marker, and its expression varies across specific regions of the brain.
• GFAP is also an essential compound of the cytoskeleton in astrocytes.
• The development of the central nervous system distinguishes astrocytes from other glial cells.
• GFAP is upregulated in astrocytes in response to most types of CNS injury and is widely used as a marker of astrocyte reactivity.

Protein Expression

• Expressed in cells lacking fibronectin.

Protein Localization

• Cytoplasm and is associated with intermediate filaments.

Figure 2: Immunocytochemistry/ Immunofluorescence Anti-GFAP antibody [EPR19996] (ab207165).
Green: GFAP, Red: MAP2, Blue: DAPI.

Isoforms & post-translational modifications

• Human (P14136): Isoforms 1-3: Predicted molecular weight 50 kDa.
• Mouse (P03995): Isoforms 1-2: Predicted molecular weight 49-50 kDa.
• Rat (P47819): Isoforms 1-2: Predicted molecular weight 49-50 kDa.
• Phosphorylated by PKN1

WB Experiment Tips

Figure 3: Western Blot- Anti-GFAP antibody [EPR1034Y] (ab68428).

Primary antibody: ab68428, diluted 1/10000.
Lane 1: Mouse cerebellum tissue lysate
Lane 2: Mouse brain tissue lysate

Predicted band size: 50 kDa
Detected band size: 50 kDa

Key Control Points

In experiments, in addition to addressing routine issues, special attention should be given to the following key control points:

Sample preparation:

1. Add a protease inhibitor cocktail to prevent degradation of target proteins.
2. Keep samples on ice throughout the entire sample preparation process.
3. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis

1. Load at least 20 μg of total protein from cell lysate or tissue homogenate.
2. When conducting a new WB experiment, include a lysis buffer as a control.

Transfer

1. We recommend staining the membrane with Ponceau S after the transfer to confirm its success.

Figure 3: Western Blot- Anti-GFAP antibody [EPR1034Y] (ab68428).

Primary antibody: ab68428, diluted 1/10000.
Lane 1: Mouse cerebellum tissue lysate
Lane 2: Mouse brain tissue lysate

Predicted band size: 50 kDa
Detected band size: 50 kDa

Key Control Points

In experiments, in addition to addressing routine issues, special attention should be given to the following key control points:

Sample preparation:

1. Add a protease inhibitor cocktail to prevent degradation of target proteins.
2. Keep samples on ice throughout the entire sample preparation process.
3. Determine the protein concentration of the samples using Bradford analysis, Lowry analysis, or BCA analysis.

Electrophoresis

1. Load at least 20 μg of total protein from cell lysate or tissue homogenate.
2. When conducting a new WB experiment, include a lysis buffer as a control.

Transfer

1. We recommend staining the membrane with Ponceau S after the transfer to confirm its success.

IHC Experiment Tips

Precautions

• Check the antibody product datasheet to confirm if it has been validated for IHC and verify the validated IHC types (e.g., formalin/PFA fixation, frozen sections). Always ensure that the tissue or cell line is from a tested species.
• For IHC-Fr experiments using perfusion-fixed tissues, continue fixation with fixative for 18-24 hours after tissue sampling; no further fixation is needed after sectioning. After sectioning, fix with 4°C pre-chilled PFA or methanol for 10 minutes for fresh tissues frozen with isopentane.
• After fixing tissues with aldehydes, refer to the antibody product datasheet for appropriate antigen retrieval methods. (For example, conducting IHC-Fr experiments with ab68428 requires heat retrieval with sodium citrate buffer at pH 6.0 for 20 minutes.)
• Always include positive and negative controls in each experiment. Positive controls ensure the efficacy of primary and secondary antibodies, while negative controls involve secondary antibodies only without primary antibodies.

Positive Controls

IHC-P: Human cerebrum tissue; Rat cerebrum tissue; Mouse cerebrum tissue

IHC-Fr: Mouse cerebrum tissue

Result examples

Figure 4: Multiplex immunohistochemistry - Anti-GFAP antibody [EPR1034Y] (ab68428)

Sample name: Human cerebellum tissue (formalin-fixed paraffin-embedded section).
Primary antibody: Anti-GFAP [EPR1034Y] (ab68428), dilution 1:250.
Antigen retrieval method: Sodium citrate antigen retrieval (pH 6.0)

Key Control Points

In an experiment, along with addressing routine issues, special attention should be given to the following key control points:

Sample fixation:

1. The optimal fixation time for samples depends on the size and type of tissue, but for most samples, it is advisable to fix with 4% PFA at room temperature for 18-24 hours.
2. Insufficient fixation can result in a higher signal at the edges of the sample and a weaker or no signal in the center.
3. Over-fixation can mask the epitope. While antigen retrieval can help overcome this masking, if the tissue is fixed for an extended period (e.g. over a weekend), there may still be no signal even after antigen retrieval.

Antigen Retrieval:

1. When conducting immunohistochemistry experiments on paraffin sections, we recommend using a pressure cooker for heat-induced antigen retrieval at 110°C for 15 minutes. After the retrieval, allow it to cool naturally and avoid placing it in cold water, as a sudden drop in temperature could cause detachment.
2. Using a scientific microwave may be preferable for tissues that are relatively brittle and prone to detachment during high-temperature and high-pressure antigen retrieval processes (such as bone and cartilage) or tissues with small cross-sections, like the sciatic nerve.
3. When conducting immunohistochemistry experiments on frozen tissue sections, if the samples have been fixed in aldehydes for 18-24 hours beforehand, it is advisable to consider using microwave short-time retrieval or enzymes for antigen retrieval. However, optimizing the enzyme concentration and retrieval time is essential to prevent damaging the tissue morphology of the sections.

Blocking:

1. If using HRP conjugate for detection, treat the sections with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity.
2. When conducting experiments using secondary antibodies conjugated with fluorescent labels, we recommend adding 1% BSA and a final concentration of 0.3 M glycine to the blocking solution to quench the autofluorescence induced by aldehyde groups.
3. Before incubating with the primary antibody, it is necessary to block with serum. Avoid choosing a blocking serum from the same host species as your primary antibody. We recommend using a serum matching the species of the secondary antibody. For example, goat serum can be used as the blocking solution if the secondary antibody is Goat Anti-Rabbit IgG H&L (HRP polymer) or Goat Anti-Mouse IgG H&L (HRP polymer).
4. If performing indirect detection with secondary antibodies, you should pay attention to the host species of the primary antibody and the species of the sample being tested. For example, when a primary antibody from a mouse is used to detect mouse tissue, non-specific staining can easily occur. Appropriate IgG blocking reagents can be added to prevent the secondary antibody from binding to endogenous IgG, thereby reducing non-specific staining.

References

• Erik Bongcam-Rudloff, Monica Nistér, Christer Betsholtz et al. Human glial fibrillary acidic protein: complementary DNA cloning, chromosome localization, and messenger RNA expression in human glioma cell lines of various phenotypes. Cancer Res(1991).51(5):1553-1560.
• Anders Lade Nielsen, Ida E Holm, Marianne Johansen et al. A new splice variant of glial fibrillary acidic protein, GFAP epsilon, interacts with the presenilin proteins. J Biol Chem. (2002).277(33):29983-29991.
  doi: 10.1074/jbc.M112121200