BAG2 Antibody

What is BAG2 and what cellular functions does it perform?

BAG2 is a 23-24 kDa protein belonging to the BAG (Bcl-2-associated athanogene) family of molecular chaperone regulators. It functions primarily as a co-chaperone for HSP70 and HSC70 chaperone proteins, acting as a nucleotide-exchange factor (NEF) that promotes the release of ADP from HSP70/HSC70, thereby triggering client/substrate protein release . BAG2 forms complexes with Hsp70 to assist in the proper folding of newly synthesized proteins and preventing their aggregation .

Importantly, BAG2 has been characterized as a negative regulator of the chaperone-associated ubiquitin ligase C terminus of Hsc70-interacting protein (CHIP), which participates in the ubiquitin-mediated proteasomal degradation of misfolded substrate proteins . BAG2 is widely expressed across human tissues, including brown adipose, heart and lung tissue, as well as in various tumor cells such as renal cell carcinoma, glioblastoma and thyroid carcinoma cells .

What applications are BAG2 antibodies suitable for in experimental research?

BAG2 antibodies have been validated for multiple experimental applications with specific methodological considerations for each:

For Western blot applications, BAG2 antibodies have successfully detected the protein in multiple human cell lines including A549 (lung carcinoma), HepG2, U-251, and U-87 MG cells, as well as in mouse and rat testis tissue samples . For immunostaining applications, methanol fixation has been shown to provide optimal results for BAG2 detection .

How should BAG2 antibodies be stored and handled to maintain optimal activity?

For optimal preservation of antibody activity, BAG2 antibodies should be stored at -20°C in aliquots to avoid repeated freeze-thaw cycles. Most commercial BAG2 antibodies are provided in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3 . Under these storage conditions, the antibodies remain stable for approximately one year after shipment.

For small volume antibodies (≤20μL), aliquoting is generally unnecessary for -20°C storage. Some preparations may contain 0.1% BSA as a stabilizer . When handling the antibody, it is advisable to keep it on ice during experimental procedures and avoid exposing it to room temperature for extended periods.

How can BAG2 antibodies be used to study stress-induced membraneless organelles?

Recent research has revealed that BAG2 marks a distinct phase-separated membraneless organelle triggered by various stressors, particularly hyperosmotic stress . Unlike stress granules and processing bodies, BAG2-containing granules lack RNA and ubiquitin, promoting client degradation in a ubiquitin-independent manner via the 20S proteasome .

To study these structures:

• Induction protocol: Subject cells to hyperosmotic stress (e.g., sucrose treatment for 2 hours) to induce BAG2 condensate formation .

• Co-localization studies: Use BAG2 antibodies (1:200 dilution for ICC/IF) alongside markers for:

  • HSP70 chaperones (positive co-localization expected)

  • 20S proteasome components (positive co-localization expected)

  • PA28 (PMSE) family members (positive co-localization expected)

  • Stress granule markers like TIA-1 (no co-localization expected)

  • P-bodies markers (no co-localization expected)

  • LAMP-1 or p62/SQSTM1 (no co-localization expected under normal conditions)

• Functional validation: When proteasome activity is inhibited, BAG2 condensates and autophagy markers traffic to aggresome-like structures, providing a control condition to validate antibody specificity .

What protocols are recommended for investigating BAG2's role in Tau protein regulation?

BAG2 has been implicated in the regulation of Tau protein, particularly in neurodegenerative conditions. To investigate this relationship:

• Cell models: Use neuronal cell lines such as SH-SY5Y expressing both Tau and BAG2 proteins. These can be created using plasmid constructs containing P2A self-cleaving peptides between fluorescent tags (e.g., mClover2-BAG2 and mRuby2-Tau) .

• Stress induction: Apply hyperosmotic stress (e.g., sucrose treatment) for 2 hours to trigger BAG2 condensate formation and subsequent Tau degradation .

• Analysis method: Perform western blotting with BAG2 antibodies (1:1000-1:4000 dilution) alongside Tau-specific antibodies such as PHF-1, MC-1, AT-8, and Tau-5 to monitor changes in Tau levels and phosphorylation states .

• Microscopy approach: For imaging studies, use immunofluorescence with BAG2 antibodies at 1:200 dilution to visualize BAG2 condensates on microtubules where they associate with Tau protein. Counterstain with microtubule markers to confirm localization .

Under stress conditions, BAG2 condensates remain associated with Tau on microtubules, providing a "safe reservoir" for Tau. Research has demonstrated that BAG2 expression under hyperosmotic stress leads to significant decreases in PHF-1/Tau-5 (3.66±2.03-fold), MC-1/Tau5 (0.69±0.63-fold), and AT8/Tau5 (1.13±0.90-fold) ratios .

How can researchers discriminate between ubiquitin-dependent and BAG2-mediated ubiquitin-independent protein degradation pathways?

BAG2 has been shown to facilitate ubiquitin-independent protein degradation via the 20S proteasome. To differentiate between these pathways:

• Experimental system: Utilize the ZsProSensor-1 vector system expressing ZsGreen with a PEST sequence (ZsGreen+PEST) to monitor ubiquitin-independent proteasome activity. Compare with a modified control construct lacking the PEST sequence (ZsGreen-PEST) .

• Co-immunoprecipitation: Use BAG2 antibodies for pull-down experiments to identify interaction partners in both degradation pathways. Western blot analysis should include:

  • BAG2 (1:1000-1:4000 dilution)

  • HSP70/HSC70 chaperones

  • 20S proteasome components

  • PA28 family activators

  • CHIP (negative correlation expected)

  • Ubiquitin (should be absent in BAG2 condensates)

• Microscopy approach: Perform immunofluorescence with BAG2 antibodies (1:200) co-stained with ubiquitin antibodies. BAG2-containing granules should lack ubiquitin, distinguishing them from typical ubiquitin-dependent degradation compartments .

This distinction is important as the 26S proteasome typically requires ubiquitination for client recognition, whereas the 20S proteasome capped by ATP- and ubiquitin-independent PA28a activator (present in BAG2 condensates) has different substrate specificity that doesn't require ubiquitination .

What are common issues with BAG2 antibody specificity and how can they be addressed?

When working with BAG2 antibodies, researchers may encounter specificity issues that can confound experimental results:

• Multiple band detection: BAG2 antibodies may detect bands at 25-27 kDa (observed molecular weight) versus the calculated 24 kDa . This discrepancy may reflect post-translational modifications. To confirm specificity:

  • Run positive control samples (A549, HepG2, U-251, or U-87 MG cell lysates)

  • Include BAG2 knockdown/knockout controls using BAG2 sgRNA or shRNA

  • Perform peptide competition assays with the immunizing peptide

• Cross-reactivity concerns: While BAG2 antibodies are typically raised against human BAG2, they may cross-react with mouse and rat BAG2 due to homology . To validate species reactivity:

  • Test antibody against lysates from multiple species

  • Confirm specificity through genetic knockdown experiments

  • When possible, use recombinant expression systems with tagged BAG2 as positive controls

• Background reduction: For cleaner Western blot results:

  • Optimize blocking conditions (5% non-fat milk or BSA)

  • Titrate primary antibody concentration (start with 1:2000 dilution)

  • Extend washing steps (4-5 times, 5 minutes each)

  • Consider reduced primary antibody incubation time (overnight at 4°C)

How can researchers effectively detect BAG2 condensates in stress response studies?

Detecting BAG2 condensates formed during stress responses requires specific experimental considerations:

• Stress induction protocol:

  • Hyperosmotic stress: 0.4-0.5M sucrose for 2 hours

  • Other effective stressors (heat shock, oxidative stress)

  • Time course analysis (condensate formation begins at ~30 minutes)

• Fixation methods:

  • Methanol fixation provides optimal results for BAG2 detection in condensates

  • Avoid paraformaldehyde fixation which may disrupt some condensate structures

  • Process samples quickly after stress induction as condensates can be dynamic

• Fluorescent tagging strategies:

  • For live-cell imaging, constructs containing fluorescently-tagged BAG2 (mClover2-BAG2, mRuby2-BAG2, or EYFP-BAG2) can be used

  • When using tagged constructs, confirm function using BAG2 mutants as controls:

    • BAG2-I160A (disrupts BAG domain function)

    • BAG2-Δ20-61 (affects condensate formation)

    • BAG2-S20E and BAG2-S20A (phosphorylation site mutants)

• Quantification methods:

  • Count condensate number per cell

  • Measure condensate size and intensity

  • Track condensate mobility and association with client proteins

Researchers should note that endogenous BAG2 condensates increase significantly after stress treatments (like sucrose exposure) and can be detected with antibody dilutions of approximately 1:200 for immunocytochemistry applications .

How can BAG2 antibodies be utilized to investigate its role in disease models?

BAG2 has been implicated in various disease processes, particularly neurodegenerative conditions and cancer. When studying disease models:

• Neurodegenerative disease models:

  • For tauopathies: Use BAG2 antibodies in conjunction with phospho-Tau markers (PHF-1, MC-1, AT-8) in cellular and animal models

  • For proteinopathies: BAG2 condensates may have therapeutic relevance similar to PA28 gamma expression, which improved motor coordination in Huntington's disease models

  • Experimental approach: Analyze BAG2 expression, localization, and condensate formation in disease versus control samples

• Cancer models:

  • BAG2 is expressed in various tumor cell types including renal cell carcinoma, glioblastoma, and thyroid carcinoma

  • In xenograft models: Use BAG2 antibodies for immunohistochemistry of tumor sections

  • For in vivo manipulation: Combine BAG2 antibody detection with genetic approaches (BAG2 shRNA, HA-tagged overexpression)

• Endometriosis model:

  • The BAG2/MDM2 relationship has been studied in endometriosis models using SCID mice with implanted human endometrial fragments

  • Detection methods: Use Western blot with BAG2 antibodies (1:1000-1:4000) to track expression changes

  • Manipulation approaches: Adenovirus-delivered overexpression (HA-MDM2 + FLAG-BAG2) or knockdown (sh-BAG2 + sh-MDM2)

What methodological approaches can reveal the interaction between BAG2 and CHIP in proteasomal degradation pathways?

BAG2 functions as a negative regulator of CHIP-mediated ubiquitin-dependent protein degradation. To investigate this regulatory relationship:

• Co-immunoprecipitation protocol:

  • Prepare cell lysates in non-denaturing buffer

  • Immunoprecipitate with BAG2 antibodies (use 2-5 μg per sample)

  • Probe western blots for CHIP, HSP70/HSC70, and client proteins

  • Include appropriate controls (IgG, input lysate, BAG2-depleted samples)

• Ubiquitination assays:

  • Treat cells with proteasome inhibitors (MG132) to accumulate ubiquitinated proteins

  • Immunoprecipitate client proteins

  • Probe for ubiquitin, BAG2, and CHIP

  • Compare ubiquitination levels in BAG2-overexpressing versus control conditions

• Client protein degradation analysis:

  • Perform cycloheximide chase experiments to track protein degradation rates

  • Compare degradation kinetics in conditions of:

    • BAG2 overexpression (expected to inhibit CHIP-mediated degradation)

    • BAG2 knockdown (expected to enhance CHIP-mediated degradation)

    • BAG2 mutant expression (BAG2-I160A affects BAG domain function)

• Microscopy approaches:

  • Use BAG2 antibodies (1:200) for immunofluorescence alongside CHIP antibodies

  • Under normal conditions: Limited co-localization expected

  • Under stress conditions: BAG2 forms condensates that exclude CHIP and ubiquitin

  • Client protein localization should be monitored simultaneously

Understanding this interaction is critical as BAG2 and CHIP represent two different degradation pathways: BAG2 promotes ubiquitin-independent degradation via the 20S proteasome, while CHIP facilitates ubiquitin-dependent degradation via the 26S proteasome .

What controls should be included when using BAG2 antibodies for experimental validation?

Proper experimental controls are essential for validating BAG2 antibody specificity and experimental outcomes:

For stress-response studies specifically, additional controls should include:

• Unstressed samples to establish baseline BAG2 localization

• Time-course samples to track condensate formation and resolution

• Alternative stress conditions to confirm specificity of response

• Co-staining with known stress granule markers (TIA-1) to confirm distinct identity of BAG2 condensates

How can researchers quantitatively analyze BAG2 expression and its effects on client protein degradation?

Quantitative analysis of BAG2 expression and function requires rigorous methodological approaches:

• Expression quantification:

  • Western blot: Normalize BAG2 signals to loading controls using densitometry

  • qRT-PCR: Use the 2–ΔΔCT method with appropriate reference genes

  • Sample calculation: Fold change = 2^(–[(CT,BAG2 – CT,ref)sample – (CT,BAG2 – CT,ref)control])

• Client protein degradation quantification:

  • For Tau degradation studies, measure the following ratios:

    • PHF-1/Tau-5

    • MC-1/Tau5

    • AT8/Tau5

  • In BAG2-expressing cells under hyperosmotic stress, expect significant decreases in these ratios (e.g., 3.66±2.03-fold decrease in PHF-1/Tau-5)

• Cell viability assessment:

  • Measure cleaved PARP as an apoptosis marker

  • In BAG2-depleted cells, expect increased cleaved PARP (66.9±4.7% increase)

  • In cells expressing BAG2 mutants, expect altered protection:

    • BAG2-Δ20-61: 66.7±9.4% increase in cleaved PARP

    • BAG2-I160A: 36.6±4.2% increase in cleaved PARP

• Statistical analysis recommendations:

  • Perform at least three independent biological replicates

  • Apply appropriate statistical tests (t-test for pairwise comparisons, ANOVA for multiple conditions)

  • Report p-values (significant findings typically p < 0.05)

  • Include error bars representing standard deviation or standard error