Name: Experimental Approaches for Biochemical Analysis of Glial Fibrillary Acidic Protein and Its Disease-associated Variants (Video) | JoVE Journal
Uploaded: 2025-11-28T14:20:00.000+00:00
Duration: 6 min 2 s
Description: Learn biochemical methods to analyze GFAP and its disease-associated variants in astrocytes. This protocol enables researchers to study filament assembly, aggregation, and post-translational modifications linked to Alexander disease.

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5 minutes

We thank Dr. Tracy Hagemann (Waisman Center, University of Wisconsin-Madison) for providing the R237H knock-in rats. The suggestion and support from Prof. Albee Messing (Waisman Center, University of Wisconsin-Madison) on this study are highly appreciated. This work was supported by grants from the Ministry of Science and Technology (111-2320-B-007-008 and 112-2320-B-007-006 to N.-H. L. and M.-D.P.). We also thank the BioTEM in the National Tsing Hua University (MOST-104-2731-M-007-002) for the use of the equipment in this study.

Animals used in this study are AxD model rats with the R237H knock-in mutation11. All animal experiments were approved by the Institutional Animal Care and Use Committee of the College of Life Sciences and Medicine at the National Tsing Hua University (NTHU IACUC Approval No. 109088 and 111060) and in accordance with the guidelines of the Agriculture Guidebook for the Care and Use of Laboratory Animals.
1. Purification of recombinant GFAP from bacteria (Figure 1)
<ol>
	<li>Transform E. coli BL21(DE3) pLysS strain with pET 23b vector containing cDNA of either WT or mutant GFAP.</li>
	<li>Culture bacteria in 1 L of Luria-Bertani medium supplemented with 100 &#181;g/mL ampicillin and 34 &#181;g/mL chloroamphenicol at 37 &#176;C to OD600 of ~0.2.</li>
	<li>Add isopropyl &#946;-D-1-thiogalactopyranoside (IPTG, 0.5 M) to a final concentration of 0.5 mM to induce GFAP expression at 37 &#176;C for 4 h.</li>
	<li>Harvest bacteria by centrifugation at 6,371 xg for 30 min at 4 &#176;C.</li>
	<li>Lyse bacterial in 50 mL of TEN buffer (20 mM Tris-HCl, pH 8, 5 mM EDTA, 150 mM NaCl) supplemented with 0.2 mM phnenylmethanesulfonyl fluoride (PMSF) by homogenization in a Dounce homogenizer.</li>
	<li>Centrifuge bacterial lysates at 22,045 xg for 20 min at 4 &#176;C, and then extract the pellet in Triton X-100 buffer (TEN with 1% (v/v) Triton X-100).</li>
	<li>After centrifugation under the same conditions, extract the resulting pellet in the high salt buffer (TEN with 1% (v/v) Triton X-100 and 1.5 M KCl).</li>
	<li>After centrifugation under the same conditions, wash the pellet with TEN buffer.</li>
	<li>Dissolve the final pellet in 10 mL of urea buffer (8 M urea, 20 mM Tris-HCl, pH 7.4, 5 mM EDTA, and 0.2 mM PMSF) by stirring for 3 h at room temperature.</li>
	<li>Centrifuge urea extracts at 80,000 xg in a tabletop ultracentrifuge at 4 &#176;C for 20 min.</li>
	<li>Add polyethyleneimine (1% (v/v)) to the urea-soluble fraction to a final concentration of 0.06% (v/v) and incubate on ice for 5 min.</li>
	<li>Centrifuge at 80,000 xg at 4 &#176;C for 20 min, and then dialyze the supernatant fraction against buffer A (6 M urea, 20 mM MES, pH 6.0, 14.4 mM &#946;-mercaptoethanol).</li>
	<li>Load dialysates onto a CM-Sepharose ion exchange column pre-equilibrated in buffer A in an NGC Chromatography System.</li>
	<li>Elute proteins with a linear gradient of 0-0.5 M NaCl in buffer A over 1 h at a flow rate of 1 mL/min.</li>
	<li>Pool GFAP-containing fractions and dialyze against buffer B (6 M urea, 10 mM Tris-HCl (pH 8.0), and 1 mM EDTA).</li>
	<li>Load dialysates onto an anion exchange column pre-equilibrated in buffer B. Elute proteins with a linear gradient of 0-0.5 M NaCl in buffer B over 1 h at a flow rate of 1 mL/min.</li>
	<li>Analyze column fractions by SDS-PAGE and Coomassie blue staining. Collect those containing purified GFAP and store at -80 &#176;C. 
	NOTE: Bacterial lysis can be facilitated by several freeze and thaw cycles. The final concentration of polyethyleneimine should not exceed 0.06% (v/v), above which proteins also precipitate. When working with hazardous materials like PMSF and &#946;-mercaptoethanol, it is critical to include clear safety guidance and instructions for hazardous waste disposal. PMSF is highly toxic and can cause irritation to the skin, eyes, and respiratory tract. Prepare PMSF solutions fresh in a fume hood and avoid direct contact. Use &#946;-mercaptoethanol in a well-ventilated area as it emits strong, toxic fumes.</li>
</ol>
2. In vitro assembly and sedimentation assay (Figure 2)
<ol>
	<li>Dilute purified WT or mutant GFAP to 0.3 mg/mL in 6 M urea in low salt buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA, and 14.4 mM &#946;-mercapethanol).</li>
	<li>Dialyze samples against the low salt buffer containing 4 M and then 2 M urea for 4 h at room temperature.</li>
	<li>Dialyze samples against the low salt buffer with no urea overnight at 4 &#176;C.</li>
	<li>Dialyze samples against assembly buffer (10 mM Tris-HCl, pH 7, 50 mM NaCl, and 14.4 mM &#946;-mercapethanol) at room temperature for 12-16 h.</li>
	<li>Transfer assembly mixtures onto the top of a sucrose cushion containing 0.2 mL of 0.85 M sucrose in assembly buffer.</li>
	<li>In the high-speed sedimentation assay, centrifuge the mixtures at 80,000 xg for 20 min at 20 &#176;C using a tabletop micro-ultracentrifuge.</li>
	<li>Concentrate unassembled GFAP in the supernatant fraction by performing methanol/chloroform precipitation15. Resuspend precipitates in 0.2 mL of Laemmli sample buffer (25 mM Tris-HCl (pH 6.8), 1% (w/v) sodium dodecyl sulfate (SDS), 12.5% (v/v) glycerol, 0.01% (w/v) bromophenol blue, and 355 mM &#946;-mercaptoethanol).</li>
	<li>Resuspend the assembled GFAP in the pellet fraction in Laemmli sample buffer.</li>
	<li>Run both supernatant and pellet fractions on 10% (w/v) SDS polyacrylamide gel electrophoresis (SDS-PAGE) gel and stain with 10 mL of Coomassie blue.</li>
	<li>In the low-speed sedimentation assay, centrifuge assembly mixtures at 3,000 xg for 5 min at 20 &#176;C using a benchtop centrifuge.</li>
	<li>Add 0.2 mL of Laemmli sample buffer to the non-aggregated forms of GFAP in the supernatant fraction.</li>
	<li>Resuspend aggregated GFAP directly in the pellet fraction in Laemmli sample buffer in a volume proportional to the supernatant fraction.</li>
	<li>Analyze both supernatant and pellet fractions by 10% (w/v) SDS-PAGE gel, followed by Coomassie blue staining. 
	NOTE: Methanol and chloroform are toxic solvents and can cause severe health effects. Avoid skin contact and inhalation. Handle with care in a fume hood to prevent spills.</li>
</ol>
3. Negative staining and electron microscopy (Figure 2)
<ol>
	<li>Glow discharge the Formvar/carbon-coated copper grids for 45 s at 20 mA in a Glow Discharge Cleaning system.</li>
	<li>Deliver assembly mixtures to the glow-discharged grid and allow samples to bind to the support film for 60 s.</li>
	<li>Remove excess liquid by wicking the edge of the grid with a piece of blotting paper.</li>
	<li>Wash grids with distilled water, then stain with 20 &#181;L of 1% (w/v) uranyl acetate for 60 s.</li>
	<li>Remove excess staining solution and allow the grid to air-dry for 30 s.</li>
	<li>Examine grids under an H-7600 transmission electron microscope (TEM) using high-resolution mode at an accelerating voltage of 100 kV. 
	NOTE: Glow-discharging the grids in a vacuum evaporator before adding the protein sample improves sample adsorption to the grid. Uranyl acetate is a radioactive and highly toxic chemical commonly used in electron microscopy for negative staining. Handling it requires strict adherence to safety protocols and hazardous waste disposal instructions to minimize exposure risks to personnel and the environment. Prepare uranyl acetate solutions only in a fume hood to prevent inhalation of airborne particles or vapors.</li>
</ol>
4. Oxidative modification (Figure 3)
<ol>
	<li>Dilute GFAP to 0.3 mg/mL in 6 M urea in low salt buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA and 14.4 mM &#946;-mercapethanol) at room temperature for 2 h.</li>
	<li>Dialyze against the low salt buffer containing 4 M urea for 2.5 h at room temperature. Stir the buffer gently using a magnetic stirrer to maintain uniform distribution and prevent concentration gradients.</li>
	<li>After 2.5 h, transfer the dialysis membrane to the low salt buffer containing 2 M urea for another 2.5 h at room temperature, ensuring gentle stirring throughout the process.</li>
	<li>Dialyze against the low salt buffer with no urea at 4 &#176;C for 18 h.</li>
	<li>Treat dialyzed samples with 10 mM H2O2 for 15 min, followed by treatment with 10 or 100 mM dithiothreitol (DTT) for an additional 15 min at room temperature.</li>
	<li>Analyze protein samples by standard 10% (w/v) SDS-PAGE without &#946;-mercapethanol and stain with 10 mL of Coomassie blue. 
	NOTE: When using H2O2, handle with caution as it is a strong oxidizing agent and can cause burns. Wear personal protective equipment to avoid contact with skin and eyes. DTT can cause irritation to the skin, eyes, and respiratory system. When working with DTT and H2O2, it is essential to follow proper safety protocols to minimize risks.</li>
</ol>
5. Purification of GFAP from the brain of AxD rats (Figure 4A,B)
<ol>
	<li>Extract brain tissues with 10 mL of TEN buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 5 mM EDTA) using a Douce homogenizer.</li>
	<li>Centrifuge the brain homogenates at 76,000 xg at 4 &#176;C for 20 min, and then extract the resulting pellet with 10 mL of Triton X-100 buffer (1% (v/v) Triton X-100 in TEN buffer).</li>
	<li>Centrifuge at 76,000 xg at 4 &#176;C for 20 min, and then extract the resulting pellet with 10 mL of sucrose buffer (0.85 M sucrose and 0.5% (v/v) Triton X-100 in TEN buffer).</li>
	<li>Centrifuge at 76,000 xg at 4 &#176;C for 20 min, and then extract the resulting pellet with 10 mL of high salt buffer (1.5 M KCl and 0.5% (v/v) Triton X-100 in TEN buffer).</li>
	<li>Centrifuge at 76,000 xg at 4 &#176;C for 20 min, and then extract the final pellet with 10 mL of urea buffer (8 M urea, 10 mM Tris-HCl, pH 7.4, and 5 mM EDTA).</li>
	<li>Collect the supernatant fraction and dialyze against the Q column buffer (6 M urea, 10 mM Tris-HCl, pH 8, 5 mM EDTA, and 14.4 mM &#946;-mercaptoethanol).</li>
	<li>Load the dialysates onto an anion exchange column in an NGC Chromatography System.</li>
	<li>Elute the bound protein with a linear gradient of 0-0.5 M NaCl in Q buffer at a flow rate of 1 mL/min.</li>
	<li>Pool GFAP-containing fraction and dialyze against S column buffer (6 M urea, 20 mM MES, pH 6, and 14.4 mM &#946;-mercaptoethanol).</li>
	<li>Apply dialysates to a cation exchange column, and then elute the bound proteins with a linear gradient of 0-1 M NaCl in S buffer at a flow rate of 1 mL/min.</li>
	<li>Analyze eluted fractions by SDS-PAGE and Coomassie blue staining, and collect those containing purified GFAP.</li>
</ol>
6. Analysis of GFAP ubiquitination by immunoblotting (Figure 4C)
<ol>
	<li>Run purified GFAP on 10% (w/v) SDS-PAGE gel.</li>
	<li>Transfer the proteins from the gel to a nitrocellulose membrane.</li>
	<li>Block the membrane by incubating it with 10 mL of 3% (w/v) bovine serum albumin in Tris-buffered saline (TBS: 20 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing 0.1% (v/v) Tween 20 (TBST) for 1 h at room temperature.</li>
	<li>Incubate the membrane with 5 &#181;L of anti-GFAP and 5 &#181;L of anti-ubiquitin primary antibodies diluted in 5 mL of TBST at 4 &#176;C overnight. Wash the membrane three times with TBST.</li>
	<li>Incubate the membrane with 5 &#181;L of secondary antibodies conjugated with fluorescent dyes, diluted in 5 mL of TBST, at room temperature for at least 1 h. Wash the membrane three times with TBST.</li>
	<li>Acquire images using a fluorescent image detection system.</li>
	<li>Analyze in vitro assembly of native GFAP by TEM and sedimentation assays (Figure 5) using the same procedure as described in steps 2.1 to 2.13 and steps 3.1 to 3.6.</li>
</ol>

The authors have nothing to disclose.

This protocol establishes a comprehensive experimental framework for the biochemical characterization of GFAP and its disease-associated mutants. By providing a reliable and standardized approach, this method is a valuable resource that enhances our understanding of the biological roles of GFAP and its pathological implications, particularly in the context of AxD. In this section, we discuss several critical aspects of the protocol, including potential modifications and troubleshooting strategies, limitations of the meth...

Alexander disease (AxD) is a genetic disorder resulting from dominant mutations in the gene that codes for glial fibrillary acidic protein (GFAP)1, a type III intermediate filament (IF) protein predominantly found in mature astrocytes. AxD is characterized by GFAP upregulation2, reactive astrogliosis3, and the accumulation of Rosenthal fibers, which are protein inclusions composed mainly of GFAP along with associated proteins such as &#945;B-crystallin4 and ubiquitin5. Mutations in the GFAP gene disrupt normal filament assembly6, leading to abnormal aggregation, induction of cellular stress responses7, and further destabilization through aberrant post-translational modifications (PTMs) such as phosphorylation8, oxidation9, and ubiquitination10.
Despite GFAP&#39;s central role in AxD pathology, the molecular mechanisms by which mutant GFAP disrupts filament assembly, promotes aggregation, and induces cellular stress remain poorly understood. This knowledge gap hinders the development of targeted therapies to address GFAP aggregation and toxicity. Studying the biochemical properties of GFAP and its AxD-associated variants is crucial for uncovering the molecular mechanisms underlying disease progression. However, the aggregation-prone property of GFAP and its altered assembly behavior in disease-associated variants present unique challenges for experimental analysis. Controlled biochemical studies require purified GFAP, obtained either from animal models or through recombinant expression, to investigate the effects of AxD mutations on filament assembly, aggregation, and pathological modifications.
The experimental approaches presented in this study offer several advantages over alternative techniques. For instance, while cell-based models11 and immunohistochemical studies12 provide valuable insights into GFAP expression and localization, they often fail to capture the precise biochemical mechanisms of filament assembly and aggregation due to the complexity of cellular environments. In contrast, the methods described here, such as in vitro filament assembly assays, sedimentation analyses, and PTM characterization, enable controlled investigation of the intrinsic biochemical properties of GFAP and its disease-associated variants. These approaches allow researchers to dissect filament assembly and aggregation behavior in a simplified system, facilitating the identification of molecular mechanisms underlying GFAP dysfunction. Previous studies have demonstrated the utility of such biochemical techniques for analyzing IF proteins, including desmin13 and keratins14, underscoring their applicability to GFAP research.
Additionally, this manuscript provides information to help readers determine whether the described methods are appropriate for their specific applications. Researchers interested in studying biochemical properties of GFAP, its assembly behavior, or PTMs will find these techniques particularly useful for investigating disease-associated variants and their pathological effects. By integrating these biochemical approaches with other experimental systems, such as cell-based studies or animal models, researchers can achieve a more comprehensive understanding of GFAP dysfunction in AxD and other related astrocytopathy.
The overall goal of this method is to present experimental approaches for the biochemical analysis of GFAP and its AxD-associated variants, including protein purification techniques, in vitro filament assembly assays, sedimentation analyses, and PTM characterization. These methods provide critical insights into the molecular basis of GFAP dysfunction in AxD, paving the way for therapeutic development. Moreover, the application of these experimental techniques will advance our understanding of GFAP biochemistry in AxD and contribute to the broader field of IF research.

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experimental approaches for biochemical analysis of glial fibrillary acidic protein and its disease-associated variants

Experimental Approaches for Biochemical Analysis of Glial Fibrillary Acidic Protein and Its Disease-associated Variants

Glial Fibrillary Acidic Protein (GFAP) is a key intermediate filament protein critical for maintaining the structural integrity and function of astrocytes in the central nervous system. Mutations in GFAP are the root cause of Alexander disease (AxD), a rare and often fatal neurodegenerative disorder characterized by elevation of GFAP levels and accumulation of GFAP in the form of Rosenthal fibers. Here, we outline a comprehensive set of experimental approaches for the biochemical characterization of GFAP and its disease-causing variants. Using optimized expression and advanced purification techniques, we achieved high yields and purity of both wild-type and mutant GFAP proteins. Biochemical assays were employed to evaluate the effects of pathogenic mutations on filament assembly, solubility, and aggregation. Additionally, we explored the role of aberrant posttranslational modifications in GFAP aggregation and their impact on filament properties. This work advances our understanding of GFAP's role in AxD and lays a foundation for developing therapeutic strategies targeting GFAP dysfunction. Furthermore, the methodologies presented here serve as valuable tools for investigating the biochemical consequences of GFAP mutations and advancing interventions for GFAP-related disorders.

To investigate how AxD-causing mutations affect GFAP assembly properties, we produced recombinant proteins in bacteria using a PET-based expression system. We detail the protocols for expressing and purifying GFAP, using WT GFAP as an example. Following IPTG induction (Figure 1A, lane 2), GFAP was purified through a three-step process involving inclusion body preparation (Figure 1B) and ion exchange chromatography with a CM column (Figure 1C...

This study outlines experimental methods to biochemically characterize GFAP and its disease-causing mutations, focusing on filament assembly, aggregation, and post-translational modification. High-purity GFAP proteins were analyzed, revealing insights into Alexander disease mechanisms that may provide a framework for studying potential therapeutic targets and understanding GFAP mutations and related disorders.

Glial Fibrillary Acidic Protein (GFAP) is a key intermediate filament protein critical for maintaining the structural integrity and function of astrocytes ...

My research focused on GFAP changes in affected astrocyte and how their dysfunction leads to Alexander disease. We share easy to follow methods to study GFAP. Disease related versions of GFAP are harder to study because they do not assemble properly, tend to clump together, and have unusual modifications.To begin, place glow discharge form bar and carbon coated copper grids in a glow discharge cleaning system. Clean the grids for 45 seconds at 20 milli amperes. Deliver the assembly mixtures onto the glottis charged grid.Remove excess liquid by wicking the edge of the grid with a piece of blotting paper. Then wash the grids with distilled water. Stain the grid with 20 microliters of 1%urinal acetate for 60 seconds.Remove the excess staining solution and allow the grid to air dry for 30 seconds. Examine the prepared grids using a transmission electron microscope in high resolution mode at an accelerating voltage of 100 kilovolts. Extract brain tissues from Alexander disease rats, using a deuce homogenizer, containing 10 milliliters of 10 buffer.Centrifuge the brain homogenates at 76, 000 G at four degrees Celsius for 20 minutes. Then sequentially extract the resulting pellet with 10 milliliters of Triton X-100 buffer, sucrose buffer, high salt buffer, and urea buffer. Collect the supernatant fraction and dialyze it against Q column buffer.Now load the dialyzed sample onto an anion exchange column in an NGC chromatography system. Elute the bound proteins using a linear gradient of zero to 0.5 molar sodium chloride in Q buffer at a flow rate of one milliliter per minute. Pool the glial fibrillary acidic protein containing fractions and dialyze them against S column buffer.Now apply the dialyzed sample to a cation exchange column and elute the bound proteins using a linear gradient of zero to one molar sodium chloride in S buffer at a flow rate of one milliliter per minute. Analyze the eluded fractions by SDS page and Coomassie blue staining. Collect those fractions containing purified glial fibrillary acidic protein.Wild type GFAP formed uniform 10 nanometer filaments in vitro, whereas the R239H mutant produced dense aggregated structures, under low speed centrifugation, most wild type GFAP remained in the supernatant, while the majority of R239H GFAP was found in the pellet fraction. Under high speed centrifugation, nearly all wild type GFAP and R239H GFAP were found in the pellet fractions confirming efficient sedimentation. Following hydrogen peroxide treatment, wild type GFAP formed multiple high molecular weight bands, which were reduced to monomers by DTT.In the absence of oxidative stress, R239H GFAP formed a high molecular weight band around 180 kilodaltons, which required high concentrations of dithiothreitol to convert to a monomeric form. Quantification confirmed that a higher proportion of R239H GFAP remained in high molecular weight forms compared to wild type GFAP. Immuno blotting confirmed that native GFAP from R237H rat brain was ubiquitinated as shown by overlapping signals for GFAP and ubiquitin.Electron microscopy revealed that GFAP from Alexander disease rat brain failed to form filaments. In low speed centrifugation, most GFAP from Alexander disease rat brains remained in the supernatant, while under high speed centrifugation, it sedimented into the pellet fraction. Quantification confirmed a shift in GFAP distribution from supernatant to pellet between low speed and high speed centrifugation.We still lack a clear picture of how mutant GFAP alters filaments, drive aggregation, and impairs cells. Our protocol directly investigate these mechanisms. We have developed a standardized method to purify both normal and mutant GFAP, making it much easier to study the protein and its role in disease.This protocol streamlines preparing normal and mutant GFAP to clarify how filament assembly, aggregation, and modifications contribute to Alexander disease.

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This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Learn biochemical methods to analyze GFAP and its disease-associated variants in astrocytes. This protocol enables researchers to study filament assembly, aggregation, and post-translational modifications linked to Alexander disease.

Experimental Approaches for Biochemical Analysis of Glial Fibrillary Acidic Protein and Its Disease-associated Variants

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