BAG1 Antibody

What is BAG1 and what cellular functions does it regulate?

BAG1 is a multifunctional protein that exists in multiple isoforms (-1L, -1M, -1S, -1XS) and serves as a co-chaperone related to the molecular chaperone HSP70/HSC70. It modulates gene transcription through mechanisms that remain incompletely defined . BAG1 interacts with multiple partners, particularly c-Raf, and has well-documented anti-apoptotic and prognostic roles in several human malignancies including breast carcinoma, hepatocellular carcinoma, leukemia, lung adenoma, neuroblastoma, and prostate cancer . The protein structure includes a ubiquitin-like domain in the middle and a BAG domain at the C-terminal region, with the BAG domain consisting of a three-helix bundle structure essential for interaction with HSP70/HSC70 .

What applications can BAG1 antibodies be used for?

BAG1 antibodies can be employed in multiple experimental applications as demonstrated with antibody 19064-1-AP:

This versatility makes BAG1 antibodies valuable tools for investigating protein expression, localization, and interactions across multiple experimental systems .

What are the expected molecular weights for BAG1 detection?

While the calculated molecular weight of BAG1 is 39 kDa, researchers should expect to observe different band patterns depending on the isoforms present in their samples. Typically, bands appear at:

• 50-55 kDa (corresponding to longer isoforms)

• 30-33 kDa (corresponding to shorter isoforms)

Understanding these patterns is crucial for accurately interpreting Western blot results, as the expression of different isoforms may vary according to cell type and physiological conditions.

How does BAG1 function in cancer cell biology?

BAG1 expression has been shown to correlate with drug resistance in several cancer types, suggesting its function as a protector of tumor cell survival upon exposure to anti-tumor drugs . At the molecular level, BAG1 interacts with Raf kinase and activates its function, which can promote cancer cell survival through the MAPK pathway . Research using HDX-MS has demonstrated the higher-order structure of BAG1S and identified a potential "druggable" site on its BAG domain .

Recent studies have used LC-MS/MS-coupled cell-free binding experiments to map the BAG-1S:c-Raf interface, uncovering a 20-amino acid-length region of BAG-1S that interacts with c-Raf. Site-directed mutagenesis experiments revealed that K149 and L156 are hot spots for BAG-1S:c-Raf interaction, and their substitutions with alanine attenuate the survival of MCF-7 cells . This information is valuable for researchers designing experiments to target the BAG1-c-Raf interaction in cancer studies.

What is the significance of BAG1 as a biomarker in breast cancer?

BAG1 has been incorporated into commercial breast cancer prognostic assays including the oncotype DX and PAM50 . Meta-analyses suggest improved outcomes with high BAG1 mRNA and high BAG1 nuclear expression by immunohistochemistry in breast cancer patients . The BAG-1L isoform specifically binds to and increases the transcriptional activity of estrogen receptor in cells, which may explain its relevance in hormone-responsive breast cancers .

• The specific isoform being detected

• Subcellular localization (nuclear vs. cytoplasmic)

• Detection method (protein vs. mRNA)

• Integration with other prognostic markers

These considerations are essential for consistent and interpretable results across studies .

How should BAG1 antibody validation be approached?

Thorough validation of BAG1 antibodies should include:

• Western blot analysis across multiple cell lines with known BAG1 expression (e.g., HeLa, MCF-7, Jurkat cells)

• Positive and negative controls, including BAG1 knockout or knockdown samples

• Peptide competition assays to confirm specificity

• Cross-validation with multiple antibodies targeting different epitopes

• Verification of subcellular localization patterns consistent with known BAG1 distribution

Researchers should be aware that BAG1 antibodies might detect multiple isoforms with different molecular weights (observed at 50-55 kDa and 30-33 kDa) . Validation experiments should account for these potential variations to ensure accurate interpretation of results.

What are the optimal conditions for BAG1 antibody applications?

For optimal results with BAG1 antibodies, researchers should consider the following application-specific conditions:

It is recommended that each antibody be titrated in the specific testing system to obtain optimal results, as the optimal conditions may be sample-dependent .

How can researchers troubleshoot BAG1 detection issues?

When encountering difficulties in BAG1 detection, consider the following troubleshooting approaches:

• For weak or absent signals in Western blot:

  • Increase antibody concentration

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

  • Optimize protein loading (30-50 μg total protein per lane)

  • Ensure complete transfer to membrane

  • Try alternative blocking agents (BSA vs. milk)

• For high background in immunohistochemistry:

  • Optimize antigen retrieval conditions

  • Reduce primary antibody concentration

  • Extend washing steps

  • Use appropriate blocking solutions

  • Consider biotin/avidin blocking if using biotin-based detection systems

• For non-specific bands in Western blot:

  • Ensure sample preparation maintains protein integrity

  • Include protease inhibitors during lysis

  • Consider the presence of multiple isoforms (50-55 kDa and 30-33 kDa bands)

  • Verify results with alternative BAG1 antibodies targeting different epitopes

These methodological adjustments can help overcome common technical challenges in BAG1 detection .

How can BAG1 antibodies be used to study protein-protein interactions?

BAG1 interacts with numerous proteins, including heat shock proteins HSP70/HSC70, Raf-1 kinase, and Bcl-2 . To study these interactions:

• Co-immunoprecipitation: Use 0.5-4.0 μg of BAG1 antibody for 1.0-3.0 mg of total protein lysate, followed by Western blot analysis of binding partners .

• Proximity ligation assays: Combine BAG1 antibody with antibodies against suspected interaction partners to visualize protein complexes in situ.

• Pull-down assays: Use recombinant BAG1 as bait to identify novel interaction partners, then confirm with co-immunoprecipitation using BAG1 antibodies.

• Mutation analysis: Introduce mutations in the BAG domain (particularly helices 2 and 3) to disrupt specific interactions, then use BAG1 antibodies to track changes in complex formation .

• Domain mapping: Recent research has mapped the BAG-1S:c-Raf interface using LC-MS/MS-coupled cell-free binding experiments, identifying a 20-amino acid-length region critical for interaction. Researchers can design similar experiments using BAG1 antibodies to investigate other protein-protein interactions .

What experimental approaches can be used to study BAG1's role in cancer?

When investigating BAG1's role in cancer, researchers can employ several experimental approaches:

• Expression correlation studies: Use BAG1 antibodies in immunohistochemistry to analyze BAG1 expression across tumor samples and correlate with clinical outcomes, as demonstrated in breast cancer studies .

• Functional studies with BAG1 inhibition: Recent research has developed a peptide derived from the BAG-1S-interacting c-Raf region that hinders BAG domain-associated partners. This peptide, engineered with a cell-penetrating peptide motif, can penetrate cells and induce apoptosis in cancer cells .

• Genetic manipulation studies: Generate BAG1 knockout cell lines using CRISPR/Cas9 technology to study the effects on cell survival, drug resistance, and signaling pathways .

• Isoform-specific functions: Design experiments to distinguish between the roles of different BAG1 isoforms (BAG-1L, BAG-1M, BAG-1S) using isoform-specific antibodies or genetic constructs.

• Therapeutic targeting: Design experiments examining the potential of targeting BAG1 interactions as a therapeutic approach, particularly in cancers with BAG1 overexpression or MAPK-driven tumors .

How is BAG1 involved in resistance to cancer therapies?

BAG1 expression has been shown to correlate with drug resistance in several cancer types, suggesting its function as a protector of tumor cell survival upon exposure to anti-tumor drugs . Recent research has advanced our understanding of this process:

• BAG-1 interacts with multiple partners, particularly with c-Raf, promoting cancer cell survival through MAPK pathway activation.

• Modulating BAG-1-associated interactions with novel inhibitors could provide benefit for cancer therapy.

• A peptide derived from the BAG-1S-interacting c-Raf region (GO-Pep) has been shown to hinder BAG domain-associated partners, penetrate cells, and induce apoptosis in cancer cells .

These findings suggest that targeting BAG1 interactions could represent a novel therapeutic approach for BAG-1-overexpressed and/or MAPK-driven tumors. Researchers can investigate this further by:

• Analyzing BAG1 expression in treatment-resistant vs. treatment-sensitive tumors

• Studying the effects of BAG1 knockdown on drug sensitivity

• Developing and testing novel BAG1 inhibitors in combination with standard therapies

• Investigating the relationship between BAG1 expression and specific resistance mechanisms

What is known about BAG1's role outside of cancer research?

Beyond cancer, BAG1 has been studied in other biological contexts:

Recent research identified BAG1 as a bradyzoite-specific protein in Toxoplasma gondii, where the BAG1 gene is involved in bradyzoite differentiation . Studies using a BAG1 knockout strain showed that while the gene is not essential for cyst formation, it influences the efficiency of cyst formation and can promote the formation of tissue cysts in vivo .

BAG1 has also been investigated in:

• Protein quality control: BAG1 is involved in degradation-mediated protein quality control systems .

• Translation regulation: BAG1 has been shown to inhibit eukaryotic translation initiation factor eIF2α de-phosphorylation .

• Stress response: As a co-chaperone for HSP70/HSC70, BAG1 plays a role in cellular stress response mechanisms.

• Immune system interactions: In the context of T. gondii infection, BAG1 immunization has been shown to induce specific T-cell responses and enhance protective immunity in mice .

These diverse functions highlight the importance of BAG1 across multiple biological systems and disease contexts.