setd3 Antibody

What is SETD3 and what are its main biological functions?

SETD3 is a member of the protein lysine methyltransferase (PKMT) family that catalyzes the addition of methyl groups to lysine residues, with a molecular weight of approximately 67 kDa . Importantly, SETD3 has been identified as the actin-specific histidine N-methyltransferase that methylates the N3 position of His73 on β-actin .

SETD3 plays multiple crucial biological roles:

• Positive regulation of DNA-damage-induced apoptosis in colon cancer cells

• Functional cross-talk with tumor suppressor p53, binding p53 in response to doxorubicin treatment

• Regulation of p53 target gene activation following DNA damage

• Muscle differentiation and smooth muscle contraction

• Female reproduction (SETD3-deficient female mice display primary dystocia and reduced litter sizes)

Experimental evidence demonstrates that SETD3 depletion from HCT-116 cells results in significant inhibition of apoptosis after doxorubicin treatment, underlining its importance in DNA damage response pathways .

What applications are SETD3 antibodies validated for in research?

SETD3 antibodies are validated for several critical applications:

For Western blotting, SETD3 typically appears as a band at approximately 67 kDa . Positive controls include MCF-7 cells for WB and mouse small intestine or liver tissue for IHC applications .

How should SETD3 antibodies be stored to maintain optimal activity?

For maximum preservation of antibody performance:

• Store at -20°C according to manufacturer recommendations

• Antibodies in glycerol buffer (like those in PBS with 0.02% sodium azide and 50% glycerol) are stable for one year after shipment when properly stored

• Some manufacturers specifically recommend against aliquoting (e.g., "Do not aliquot the antibody")

• For certain formulations (e.g., 20μl sizes), the presence of 0.1% BSA may help stabilize the antibody

Proper storage maintains specificity and sensitivity for detecting endogenous SETD3 in human and monkey samples as validated by manufacturers .

What species reactivity has been confirmed for commercial SETD3 antibodies?

Commercial SETD3 antibodies show reactivity with:

When selecting an antibody, verify species reactivity to ensure compatibility with your experimental model system .

What is the importance of SETD3 as a histidine methyltransferase?

SETD3's identification as the actin-specific histidine N-methyltransferase represents a significant discovery in protein post-translational modifications . Key points include:

• SETD3 methylates β-actin specifically at His73, a modification conserved across evolution

• This represents a rare case of histidine methylation, as protein histidine methylation is an uncommon post-translational modification

• SETD3 has very high affinity for both SAM (KM≈0.3 μM) and β-actin (KM≈0.8-3 μM)

• SETD3 catalytic activity is relatively slow, with kcat values of 0.6-0.8 min−1, typical of protein methyltransferases rather than small-molecule methyltransferases

The physiological significance of this activity is demonstrated by SETD3-knockout studies showing that over 90% of actin's His73 residues are methylated in wildtype cells but not in SETD3-deficient cells .

How can researchers validate SETD3 antibody specificity using knockout models?

Rigorous validation requires multiple complementary approaches:

• Generate SETD3-knockout cell lines using CRISPR/Cas9 technology

  • Multiple independent clonal knockout lines should be established (e.g., KO-A1, KO-A3, KO-A5 as described in literature)

  • Confirm genomic modifications by DNA sequencing to verify frameshift mutations or premature stop codons

• Perform Western blot analysis comparing wildtype and knockout samples

  • Absence of the 67 kDa SETD3 band in knockout lines confirms antibody specificity

  • Include positive controls (e.g., wildtype cells) and loading controls

• Functional validation through enzymatic assays

  • Mass spectrometry analysis of actin methylation status

  • In published studies, "methylation of the H73 residue was detected in almost all H73-containing β-actin peptides derived from the wildtype HAP1 cells, whereas close to 90% of the same β-actin peptides derived from Setd3-deficient cell lines were not methylated at H73"

• Complementation experiments

  • Reintroduce wildtype SETD3 into knockout cells to restore function

  • Use both wildtype and catalytically inactive SETD3 (Y313A) for mechanistic studies

What are optimal experimental designs for investigating SETD3's role in p53-dependent apoptosis?

For robust investigation of SETD3's role in apoptosis:

• Loss-of-function approaches:

  • Generate SETD3 knockout cells using CRISPR/Cas9 technology

  • Treat cells with DNA-damaging agents (doxorubicin, etoposide, or abiplatin)

  • Measure apoptosis markers (e.g., Annexin V staining, PARP cleavage)

  • Analyze expression of p53 target genes (BAX, PUMA, NOXA) by qRT-PCR

• Protein interaction studies:

  • Co-immunoprecipitation of endogenous SETD3 and p53 after DNA damage treatment

  • ChIP experiments to assess p53 recruitment to target genes with/without SETD3

  • Controls should include IgG immunoprecipitation and input samples

• Mechanistic investigation:

  • Rescue experiments comparing wildtype SETD3 vs catalytically inactive SETD3 Y313A

  • ChIP experiments reveal "SETD3 catalytic activity is required for the recruitment of p53 to its target genes"

  • In vitro binding assays (e.g., ELISA) to test direct interaction between recombinant SETD3 and p53

• Clinical correlation:

  • Kaplan-Meier survival analysis of cancer patient cohorts stratified by SETD3 expression

  • Research demonstrates that "low expression of SETD3 is a reliable predictor of poor survival" in colon cancer patients

How can researchers differentiate between SETD3's histidine methyltransferase activity and other potential functions?

To distinguish between different SETD3 functions:

• In vitro enzymatic assays:

  • Compare methylation of histidine-containing substrates (β-actin, peptide H: YPIEHGIVT) with lysine-containing substrates (histones)

  • Use mass spectrometry to identify precise methylation sites

  • Employ deuterated SAM ([²H]SAM) to track methyl group transfer

  • Test substrate specificity using histidine mimics and analogs

• Structure-function analysis:

  • Generate catalytic mutants (Y313A) that abolish methyltransferase activity

  • Compare phenotypes between knockout and catalytic mutant rescue experiments

  • Research shows that "SETD3 catalytic activity is required for the recruitment of p53 to its target genes"

• Substrate specificity investigation:

  • Test recombinant SETD3 against peptide libraries containing histidine or histidine analogs

  • Recent research has shown that "SETD3 has a broader substrate scope beyond histidine, including N-nucleophiles on the aromatic and aliphatic side chains"

  • Compare activity on wildtype peptides versus H73A mutant peptides

What controls are critical when studying SETD3's interaction with actin?

Essential controls for SETD3-actin interaction studies include:

• Substrate controls:

  • Wildtype β-actin vs. H73A mutant β-actin (no methylation occurs with H73A)

  • Native vs. denatured/refolded actin (research shows that "yeast-produced human β-actin became a good substrate for SETD3 when purified in denaturing conditions and re-folded into nucleotide-free quasi-native actin")

  • Actin-derived peptides (peptide H: YPIEHGIVT) vs. modified peptides (peptide A: YPIEAGIVT)

  • Nucleotide-bound states (ATP-β-actin or ADP-β-actin showed no methylation by SETD3)

• Enzyme controls:

  • Wildtype SETD3 vs. catalytically inactive SETD3 (Y313A)

  • SETD3 from different species (rat vs. human SETD3 show different kinetic parameters)

• Interaction controls:

  • Test actin complexed with binding partners (profilin, cofilin)

  • Research shows "methylation of profilin-β-actin was negligible and that of cofilin-β-actin complex was low"

• Detection methods:

  • Mass spectrometry to identify specific methylation sites

  • Radiochemical methylation assays with [³H]SAM or [²H]SAM

  • Compare peptide vs. protein methylation rates (actin protein is methylated ≈250-fold more efficiently than peptide H)

How should researchers interpret contradictory findings about SETD3's role in different cancer types?

When analyzing SETD3's complex roles across cancer types:

• Consider cancer-type specificity:

  • High SETD3 expression correlates with better survival in colon cancer patients

  • In breast cancer, higher SETD3 expression is associated with increased survival in general subtypes but poor survival in triple-negative and p53 mutant tumors

  • High expression of SETD3 displays oncogenic properties in lymphoma

  • In liver cancer, upregulation of SETD3 is associated with cancer development

  • Low SETD3 expression correlates with shorter disease-free survival in renal cancer

• Examine molecular context:

  • SETD3's function may depend on p53 status (functional cross-talk between SETD3 and p53)

  • Different splice variants may exist (e.g., SETD3 lacking the SET domain has different properties)

  • Consider protein-protein interactions specific to each cancer type

• Integrate functional studies with clinical data:

  • Combine in vitro findings with patient survival analysis

  • Use multiple cohorts to validate findings, as demonstrated in studies using "two-independent cohorts of colon cancer patients"

  • Consider multivariate analysis accounting for clinical covariates

• Methodological considerations:

  • Use antibodies validated for the specific application (WB, IHC)

  • Include proper controls (tissue-specific positive controls)

  • Consider methyltransferase-dependent and -independent functions

What methodological approaches can quantify SETD3's enzymatic activity?

For accurate measurement of SETD3's catalytic activity:

• In vitro methyltransferase assays:

  • Radiochemical assays using [³H]SAM or [²H]SAM to directly measure methyl transfer

  • Mass spectrometry to identify and quantify methylation sites

  • Enzyme kinetics experiments measuring KM and kcat for both SAM and protein substrates

• Kinetic parameter determination:

  • Human SETD3 shows KM ≈0.8 μM for β-actin, compared to rat SETD3's KM ≈3 μM

  • Both enzymes show high affinity for SAM (KM ≈0.3 μM)

  • Catalytic efficiencies (kcat/KM) can be calculated and compared across substrates and enzyme variants

• Substrate specificity analysis:

  • Compare activity on full-length actin versus actin-derived peptides

  • Test various histidine-containing peptides or histidine mimics

  • Research shows actin peptide H is methylated with extremely low efficiency compared to intact actin (≈0.02 nmol·min⁻¹·mg⁻¹ vs. ≈5 nmol·min⁻¹·mg⁻¹)

• Cellular methylation assays:

  • Compare methylation status in wildtype versus SETD3-knockout cells

  • Quantify methylation stoichiometry by mass spectrometry

  • Research demonstrates that methylation of H73 is detected in "almost all H73-containing β-actin peptides derived from the wildtype HAP1 cells" but not in SETD3-knockout cells

How can SETD3 antibodies be applied to study its role in enterovirus pathogenesis?

Recent research has revealed that SETD3 plays a critical role in enterovirus infection:

• Genome-scale CRISPR-Cas9 knockout screens identified SETD3 as critically important for enterovirus pathogenesis

• This involves a novel interaction between viral 2A protein and host SETD3, independent of SETD3's methyltransferase activity

• Research approaches should include:

  • Co-immunoprecipitation using SETD3 antibodies to study viral protein interactions

  • Infection experiments comparing wildtype and SETD3-knockout cells

  • Distinguishing between methyltransferase-dependent and -independent functions

What considerations apply when investigating SETD3's broader substrate specificity?

Recent discoveries show SETD3 has wider substrate scope than initially thought:

• SETD3 can methylate structurally diverse histidine mimics in actin peptides

• Activity extends beyond histidine to include "N-nucleophiles on the aromatic and aliphatic side chains"

• Experimental approaches should include:

  • Synthetic peptide libraries with histidine analogs

  • Biostructural analyses to understand substrate binding

  • Computational approaches to predict potential novel substrates

  • Mass spectrometry to identify methylation sites in cellular proteins

This expanded substrate recognition capability suggests SETD3 may have additional physiological targets beyond actin His73 methylation that remain to be discovered .