ALKBH7 Antibody

What is ALKBH7 and what cellular functions does it regulate?

ALKBH7 (Alkylated DNA repair protein alkB homolog 7) is a member of the alkB family, also known as ABH7 or SPATA11. It functions as a dioxygenase that requires molecular oxygen, alpha-ketoglutarate, and iron for its activity. ALKBH7 plays a pivotal role in programmed necrosis triggered by DNA-damaging agents through mechanisms involving mitochondrial membrane potential collapse and large-scale loss of mitochondrial function, ultimately leading to energy depletion and cellular demise . Additionally, ALKBH7 is involved in the regulation of glyoxal metabolism, with ALKBH7 deficiency offering protection against necrosis and cardiac ischemia-reperfusion injury .

What is the expression pattern of ALKBH7 across different tissues?

Quantitative real-time PCR has detected variable ALKBH7 expression across multiple tissues. The highest expression levels have been observed in the pancreas, followed by spleen, prostate, ovary, and placenta. In total, ALKBH7 expression has been detected across all 16 tissues examined in previous studies . The widespread expression pattern suggests ALKBH7 may have fundamental roles in cellular physiology beyond specific tissue functions.

What are the key specifications of commercially available ALKBH7 antibodies?

The most commonly cited ALKBH7 antibody (15470-1-AP) has the following specifications:

This antibody has been validated for Western blot and immunohistochemistry applications, with positive detection in human testis tissue .

How should ALKBH7 antibody be optimized for immunohistochemistry applications?

For optimal immunohistochemistry (IHC) results with ALKBH7 antibody, the following protocol modifications are recommended:

• Antigen retrieval should be performed with TE buffer pH 9.0, though citrate buffer pH 6.0 may serve as an alternative.

• The recommended dilution range for IHC is 1:20-1:200, but this should be titrated for each specific testing system.

• Positive controls should include human testis tissue, where ALKBH7 expression has been reliably detected.

• When developing the signal, extend chromogen development time for detecting low-abundance expression, as ALKBH7 may display variable expression levels across different tissues .

What experimental approaches can validate ALKBH7 knockdown or knockout models?

To confirm successful ALKBH7 depletion in experimental models, researchers have employed the following validation strategies:

• Western blot analysis using specific ALKBH7 antibodies to confirm protein depletion.

• Rescue experiments by re-expressing ALKBH7 in depleted cell lines to reverse phenotypes. For example, ALKBH7-depleted cells that display resistance to MMS (methyl methanesulfonate) treatment show restored sensitivity when ALKBH7 expression is reintroduced via transfection .

• Functional assays assessing mitochondrial membrane potential, as ALKBH7-depleted cells maintain their mitochondrial membrane potential after exposure to alkylating and oxidizing agents, unlike wild-type cells .

• Measurements of cellular NAD and ATP levels following DNA damage, as ALKBH7-depleted cells show rapid recovery of these metabolites compared to wild-type cells .

What are the common pitfalls when using ALKBH7 antibodies and how can they be avoided?

When working with ALKBH7 antibodies, researchers frequently encounter the following challenges:

• Non-specific binding: To minimize background, implement more stringent blocking protocols using 5% BSA or milk in TBST and extend blocking time to 2 hours at room temperature.

• Variable signal intensity: ALKBH7 expression levels vary significantly across tissues. When analyzing tissues with potentially low expression, consider using signal amplification methods or increasing the antibody concentration gradually.

• Cross-reactivity concerns: Given that ALKBH7 belongs to a family with similar proteins, validate specificity by including appropriate controls such as ALKBH7 knockout samples or peptide competition assays.

• Degradation issues: Store antibody aliquots at -20°C to maintain stability for up to one year after shipment. For 20μl size preparations containing 0.1% BSA, aliquoting is not necessary for -20°C storage .

How can researchers optimize Western blot protocols for ALKBH7 detection?

For optimal Western blot results when detecting ALKBH7:

• Use a dilution range of 1:500-1:1000 for the primary antibody .

• Given the 25 kDa size of ALKBH7, use 12-15% polyacrylamide gels for optimal resolution.

• Transfer proteins to nitrocellulose or PVDF membranes using standard wet transfer systems.

• When analyzing tissues with variable ALKBH7 expression, human testis tissue can serve as a reliable positive control .

• Consider using gradient gels if analyzing post-translational modifications that might alter the protein's migration pattern, as ALKBH7 may undergo auto-hydroxylation or other modifications .

How does ALKBH7 expression correlate with cancer prognosis and immune markers?

Analysis of ALKBH7 expression across multiple cancer types reveals significant correlations with prognostic indicators and immune system components:

• ALKBH7 shows elevated expression in 17 cancer types and reduced expression in 5 cancer types compared to paired normal tissues .

• Expression correlates significantly with tumor mutation burden (TMB) in 7 cancer types and microsatellite instability (MSI) in 13 cancer types .

• Notable correlations exist between ALKBH7 expression and tumor stemness indices, including mDNAsi (12 cancer types) and mRNAsi (13 cancer types) .

• ALKBH7 expression demonstrates significant associations with immune checkpoint genes across multiple cancers, predominantly showing negative correlations .

These findings suggest ALKBH7 may serve as a potential pan-cancer prognostic biomarker with implications for immunotherapeutic response prediction.

What is known about ALKBH7's role in immune cell infiltration across cancer types?

ALKBH7 demonstrates significant correlations with infiltrating immune cells across 31 cancer types, with particularly strong relationships observed for:

• Dendritic cells in 8 cancer types

• Macrophages in 9 cancer types

• Neutrophils in 11 cancer types

• CD8+ T cells in 14 cancer types

• B cells in 8 cancer types

• CD4+ T cells in 3 cancer types

The strongest correlations with immune infiltration have been observed in pancreatic adenocarcinoma (PAAD), prostate adenocarcinoma (PRAD), and thyroid carcinoma (THCA) . Researchers investigating these cancer types should consider ALKBH7's potential role in modulating the tumor immune microenvironment when designing experiments.

How does ALKBH7 contribute to programmed necrosis and what methodologies can investigate this mechanism?

ALKBH7 plays a crucial role in programmed necrosis following DNA damage. The following experimental approaches have been used to elucidate this mechanism:

• Cell viability assays: ALKBH7-depleted cells show resistance to cell death induced by alkylating and oxidizing agents compared to control cells .

• Mitochondrial membrane potential measurements: ALKBH7-depleted cells maintain their mitochondrial membrane potential after DNA damage, unlike wild-type cells that exhibit collapse .

• PARP activation analysis: Both wild-type and ALKBH7-depleted cells undergo similar PARP hyperactivation and initial NAD depletion after DNA damage, but only ALKBH7-depleted cells rapidly recover NAD and ATP levels .

• Metabolic profiling: Analysis of glyoxal metabolism reveals that ALKBH7-deficient systems show rewiring of glucose and glyoxal metabolism under glycative stress conditions .

These methodologies collectively demonstrate ALKBH7's critical role in mediating the metabolic collapse that drives necrotic cell death following severe DNA damage.

What are promising research areas for ALKBH7 beyond current applications?

Several emerging research directions for ALKBH7 warrant further investigation:

• Therapeutic targeting: Development of selective ALKBH7 inhibitors could potentially prevent necrosis in ischemia-reperfusion injury and other conditions where programmed necrosis contributes to pathology .

• Cancer immunotherapy biomarker: Given ALKBH7's correlations with immune checkpoint genes and infiltrating immune cells, its potential as a predictive biomarker for immunotherapy response should be explored across various cancer types .

• Metabolic regulation: Further investigation into ALKBH7's role in glyoxal metabolism could reveal novel metabolic dependencies in cancer and other diseases .

• Post-translational modifications: Exploration of ALKBH7's hydroxylase activity and potential targets could uncover new regulatory mechanisms in cellular metabolism and stress response .

Researchers entering the ALKBH7 field should consider these promising directions when designing long-term research programs.