HSF2 Antibody

What is HSF2 and why is it important in research?

HSF2 (Heat shock transcription factor 2) is a transcription factor that binds specifically to heat shock elements (HSEs) in the promoter regions of heat shock genes. Unlike HSF1, which primarily responds to classical stress inducers (heat shock, amino acid analogs, heavy metals), HSF2 is activated during early embryogenesis, spermatogenesis, and erythroid differentiation . HSF2 mediates Hsp70i bookmarking by binding to promoters in mitotic cells, recruiting protein phosphatase-2A, and interacting with the CAPG subunit of condensin enzyme to prevent chromatin compaction . Studies with HSF2-null mice revealed brain abnormalities and meiotic/gametogenic defects in both genders, indicating its crucial developmental role .

What is the molecular weight of HSF2 detected by antibodies?

HSF2 is typically detected at approximately 60 kDa on Western blots under reducing conditions . The calculated molecular weight based on amino acid sequence is 79 kDa , but the observed molecular weight is consistently around 60 kDa across multiple antibody sources . This difference between calculated and observed weight should be considered when validating antibody specificity.

What species reactivity do HSF2 antibodies typically have?

Most commercially available HSF2 antibodies react with human, mouse, and rat samples . Some antibodies show broader reactivity; for example, the HSF2 monoclonal antibody (3E2) from Enzo reportedly reacts with bovine, dog, guinea pig, hamster, human, monkey, mouse, porcine, rabbit, rat, and sheep samples . When selecting an antibody, confirm species reactivity in the product documentation, especially for less common research models.

What applications can HSF2 antibodies be used for?

HSF2 antibodies have been validated for multiple applications:

Optimal dilutions should be determined empirically for each specific antibody and experimental system .

How should HSF2 antibodies be stored and handled?

For optimal performance and longevity:

• Store lyophilized antibodies at -20°C upon receipt .

• After reconstitution, store at 4°C for short-term use (up to one month) or aliquot and store at -20°C to -70°C for long-term storage (six months) .

• Avoid repeated freeze-thaw cycles which can degrade antibody performance .

• Some formulations contain glycerol (typically 50%) and sodium azide (0.02-0.09%) as preservatives .

• For antibodies stored in PBS with glycerol, aliquoting is not necessary for -20°C storage in some cases .

How can I optimize HSF2 antibody dilutions for different applications?

Optimization strategies include:

• Western Blot: Begin with manufacturer's recommended dilution (typically 0.5-1 μg/ml) and adjust based on signal intensity. Use PVDF membrane and appropriate blocking buffer (e.g., Immunoblot Buffer Group 3 as used with R&D Systems antibody) .

• Immunohistochemistry: For paraffin sections, test both citrate buffer (pH 6.0) and TE buffer (pH 9.0) for antigen retrieval . Titrate antibody concentration between 1:250-1:1000 for polyclonal antibodies .

• Immunofluorescence: For fixed cells, perform enzyme antigen retrieval (e.g., using IHC enzyme antigen retrieval reagent) . Block with 10% normal serum from the same species as the secondary antibody .

• Flow Cytometry: For intracellular HSF2 detection, fix cells with 4% paraformaldehyde and permeabilize before antibody incubation. Use appropriate isotype control antibodies to assess non-specific binding .

What are the differences between monoclonal and polyclonal HSF2 antibodies?

Select based on your specific application needs and whether epitope conservation across species is important for your research.

How can I validate the specificity of an HSF2 antibody in my experimental system?

Comprehensive validation should include:

• Molecular weight verification: Confirm the band appears at ~60 kDa in Western blots .

• Positive controls: Use cell lines known to express HSF2, such as HeLa, 293T, NIH-3T3, HepG2, Jurkat, or A549 cells .

• Negative controls/knockdown: Compare with HSF2 knockout or knockdown samples using CRISPR/Cas9 or siRNA technology. Several suppliers offer HSF2 CRISPR knockout plasmids that can be used to generate control cells .

• Immunogen competition: Pre-incubate the antibody with the immunizing peptide before application to confirm specificity.

• Cross-reactivity assessment: Particularly important if studying both HSF1 and HSF2, as they share homology. Use antibodies raised against unique regions to avoid cross-reactivity .

How can HSF2 antibodies be used to study HSF2 activation during stress and development?

HSF2 undergoes dynamic changes during activation that can be studied using:

• Subcellular localization: Use immunofluorescence to track HSF2 translocation from cytoplasm to nucleus during activation . In quiescent cells, HSF2 appears as a cytoplasmic homodimer, but upon stress or differentiation signals, it trimerizes and translocates to the nucleus .

• Trimerization analysis: Use non-denaturing gel electrophoresis followed by Western blotting to detect the transition from dimers to trimers.

• ChIP assays: Study HSF2 binding to heat shock elements in target gene promoters using ChIP with HSF2-specific antibodies. The antibody from R&D Systems (AF5227) has been validated for ChIP applications in published studies .

• Developmental timing: Track HSF2 expression during embryogenesis, particularly in all three embryonic layers at day 7.5 and the head fold at day 8.5 in mouse models .

• Co-immunoprecipitation: Study HSF2 interactions with HSF1 or other proteins using antibodies suitable for immunoprecipitation .

What controls should be included when studying HSF2 in specific tissues like testis and brain?

When studying tissue-specific functions of HSF2:

• Tissue-specific expression controls: For testis studies, note that HSF2 is expressed in spermatocytes and spermatogonia, but not in elongated spermatids, spermatozoa, or Sertoli cells . Include these cell types as internal positive and negative controls.

• Developmental timing: For brain studies, include appropriate developmental stage controls, as HSF2-null mice show brain abnormalities .

• Comparative analysis: When studying both HSF1 and HSF2, use antibodies that can distinguish between these related factors, such as the 10H8 (for HSF1) and 3E2 (for HSF2) antibodies described in the Northwestern University technology .

• RNA validation: Complement protein detection with RNA analysis (RT-PCR or RNA-seq) to confirm expression patterns.

• Functional validation: Consider using HSF2 knockout models or targeted disruption to verify the specificity of observed phenotypes .

How can HSF2 antibodies be used to study the role of HSF2 in cancer?

Recent research has implicated HSF2 in cancer biology, particularly in hepatocellular carcinoma where it regulates aerobic glycolysis by suppressing FBP1 . To study HSF2 in cancer:

• Expression analysis: Compare HSF2 levels between normal and tumor tissues using immunohistochemistry with antibodies like HPA031455 or PA1607 .

• Functional studies: Use HSF2 antibodies in combination with CRISPR/Cas9-mediated knockout or activation systems available from vendors like Santa Cruz Biotechnology to assess the functional consequences of HSF2 modulation in cancer cell lines.

• Pathway analysis: Investigate HSF2 interactions with other proteins in cancer pathways using co-immunoprecipitation with antibodies suitable for IP, such as sc-13517 or sc-74529 .

• Chromatin dynamics: Study HSF2's role in chromatin remodeling during cancer progression using ChIP-seq approaches with ChIP-certified antibodies like those from Atlas Antibodies .

What methods can be used to study HSF2 interaction with other heat shock factors?

To investigate HSF2 interactions with other HSF family members:

• Co-immunoprecipitation: Use antibodies specific to HSF2 (e.g., 3E2) to pull down HSF2 and associated proteins, then probe for HSF1 or other partners.

• Proximity ligation assay (PLA): Detect in situ protein-protein interactions between HSF2 and other factors using antibodies from different host species.

• Bimolecular fluorescence complementation (BiFC): Study direct interactions by expressing HSF2 and potential partners fused to complementary fragments of a fluorescent protein.

• Dual immunofluorescence: Use compatible HSF1 and HSF2 antibodies to study co-localization during stress response or development.

• Sequential ChIP (Re-ChIP): Determine if HSF1 and HSF2 co-occupy the same genomic regions using sequential immunoprecipitation with antibodies against both factors.