ALKBH5 Antibody

What is ALKBH5 and why is it significant in epitranscriptomic research?

ALKBH5 (alkB homolog 5) is a critical RNA demethylase that specifically removes N6-methyladenosine (m6A) modifications from mRNAs. It functions as an "eraser" in the dynamic regulation of m6A, a prevalent RNA modification in eukaryotes. ALKBH5 is a protein of approximately 44.3 kilodaltons with 458 amino acids, though its observed molecular weight in experimental conditions typically ranges between 40-50 kDa .

The significance of ALKBH5 in epitranscriptomic research stems from its crucial roles in:

• RNA metabolism and stability

• Stem cell maintenance and differentiation

• Cancer progression or suppression in a context-dependent manner

• Immune response modulation

Recent studies have demonstrated ALKBH5's involvement in maintaining hematopoietic stem cell self-renewal capacity, tumor suppression in bladder cancer and hepatocellular carcinoma, and promotion of angiogenesis in glioblastoma .

How should I select the appropriate ALKBH5 antibody for my specific research application?

Selection criteria should be based on:

Always verify:

• Whether the antibody has been validated for your specific application (WB, IHC, IF, RIP)

• Cross-reactivity with your species of interest

• Published literature using the antibody for similar experiments

• Evidence of validation through knockdown/knockout controls

For instance, antibody catalog #16837-1-AP has been validated for multiple applications including WB (1:2000-1:10000 dilution), IHC (1:50-1:500 dilution), and has confirmed reactivity with human, mouse, and rat samples .

What are the optimal conditions for ALKBH5 detection by Western blotting?

Sample Preparation:

• Extract proteins using RIPA buffer with protease inhibitors

• Load 20-50 μg of total protein per lane

• Include phosphatase inhibitors if studying post-translational modifications

Electrophoresis and Transfer:

• Use 10-12% SDS-PAGE gels for optimal separation

• Transfer to PVDF membrane at 100V for 60-90 minutes or 30V overnight at 4°C

Antibody Incubation:

• Primary antibody dilution: 1:2000 to 1:10000 depending on antibody sensitivity

• Incubate overnight at 4°C or for 1.5 hours at room temperature

• Secondary antibody typically at 1:5000-1:10000 dilution

Detection Parameters:

• Expected band size: 40-50 kDa (main band)

• Possible additional bands at ~52 kDa (isoform)

• Use GAPDH (36 kDa) as a loading control

Validation Controls:

• Include positive control samples (HeLa cells, HEK-293 cells show good expression)

• Include negative control (siRNA knockdown) to confirm specificity

Published studies have successfully used ALKBH5 antibodies for Western blotting to detect expression in bladder cancer cell lines (T24 and 5637) and various other cell types including hematopoietic stem cells .

How can I optimize ALKBH5 antibody protocols for immunohistochemistry?

Tissue Preparation:

• Fix tissues in 10% neutral buffered formalin

• Use paraffin embedding for most applications

• Cut sections at 4-5 μm thickness

Antigen Retrieval:

• TE buffer pH 9.0 is recommended as the primary method

• Alternative: citrate buffer pH 6.0 (may be less effective for some antibodies)

• Heat-induced epitope retrieval: 95-98°C for 15-20 minutes

Antibody Parameters:

• Dilution range: 1:50-1:500 for polyclonal (#16837-1-AP)

• Dilution range: 1:500-1:2000 for monoclonal (#67811-1-Ig)

• Incubation: overnight at 4°C or 1-2 hours at room temperature

Detection Systems:

• Use HRP-conjugated secondary antibodies with DAB chromogen

• Alternative: fluorescent-labeled secondary antibodies for co-localization studies

Controls and Validation:

• Positive control tissues: testis and brain tissues show reliable expression

• Negative controls: primary antibody omission

• Validation: comparison with RNA expression data

Special Considerations:

• Human testis, lung cancer, and mouse testis tissues have been successfully used as positive controls

• Signal localization should be primarily nuclear with some cytoplasmic staining

What methods should I employ to validate ALKBH5 antibody specificity?

A comprehensive validation approach should include:

Genetic Validation:

• siRNA/shRNA knockdown: Multiple siRNA constructs targeting different regions of ALKBH5

• CRISPR/Cas9 knockout: Complete elimination of target protein

• Overexpression: Ectopic expression in low-expressing cell lines

Analytical Validation:

• Multiple antibodies: Test different antibodies recognizing distinct epitopes

• Peptide competition assay: Pre-incubation with immunizing peptide

• Molecular weight confirmation: Compare observed MW with predicted size

• Multiple detection methods: Compare results from WB, IHC, and IF

Experimental Validation:

• Species cross-reactivity testing

• Cell/tissue type diversity testing

• Lot-to-lot consistency evaluation

Research has shown that knockdown validation is critical, as some antibodies may detect non-specific bands. For example, one researcher reported that while a certain ALKBH5 antibody failed to show knockdown with siRNA constructs, the Cell Signaling antibody (#80283) clearly demonstrated knockdown in the same lysates .

Why might I observe multiple bands or inconsistent molecular weights when detecting ALKBH5 by Western blot?

Multiple bands or varying molecular weights can occur due to:

Biological Factors:

• Protein isoforms: ALKBH5 has reported isoforms of 40-44 kDa and 52 kDa

• Post-translational modifications: Phosphorylation, ubiquitination

• Proteolytic processing: Partial degradation during sample preparation

• Alternative splicing variants: Tissue-specific expression

Technical Factors:

• Sample preparation issues: Protein degradation during extraction

• Incomplete denaturation: Improper sample heating or SDS concentration

• Non-specific binding: Insufficient blocking or antibody cross-reactivity

• Transfer problems: Incomplete transfer of higher MW proteins

Troubleshooting Approaches:

• Use fresh samples and protease inhibitors

• Optimize denaturation conditions (95°C for 5 minutes with fresh β-mercaptoethanol)

• Increase blocking time and wash stringency

• Use gradient gels for better resolution of potential isoforms

• Validate with knockdown controls to confirm which bands are specific

Published studies report the main ALKBH5 band between 40-50 kDa, with some variation depending on cell type and experimental conditions .

How should I interpret conflicting results between ALKBH5 expression and function in different cancer types?

ALKBH5 demonstrates context-dependent roles across different cancers, requiring careful interpretation:

Tumor Suppressor Role:

• Bladder cancer: ALKBH5 inhibits proliferation and increases cisplatin sensitivity

• Hepatocellular carcinoma: ALKBH5 is downregulated and associated with worse prognosis

Oncogenic Role:

• Glioblastoma: ALKBH5 promotes angiogenesis

• Colorectal cancer: ALKBH5 drives immune suppression via the AXIN2-Wnt-DKK1 axis

Interpretation Framework:

• Confirm ALKBH5 expression level by multiple methods (WB, qPCR, IHC)

• Assess m6A levels in conjunction with ALKBH5 expression

• Identify tissue-specific downstream targets (e.g., CK2α in bladder cancer, AXIN2 in colorectal cancer)

• Consider tissue context and tumor microenvironment

• Evaluate experimental models (cell lines vs. patient samples vs. animal models)

Reconciliation Strategies:

• Perform mechanism studies to identify tissue-specific targets

• Use rescue experiments to confirm direct causality

• Evaluate temporal dynamics of expression during disease progression

• Consider dual roles based on subcellular localization or interaction partners

How can ALKBH5 antibodies be utilized in RNA immunoprecipitation (RIP) experiments to study m6A modification?

RIP using ALKBH5 antibodies allows the identification of ALKBH5-bound RNAs and assessment of regulatory mechanisms:

Protocol Optimization:

• Crosslinking: Use formaldehyde (1%) for 10 minutes at room temperature

• Lysis buffer: Include RNase inhibitors and protease inhibitors

• Antibody amount: 5-10 μg per IP reaction

• Pre-clearing: Use protein A/G beads before adding antibody

• Controls: IgG control and input RNA essential

Analysis Methods:

• qRT-PCR: For known target validation

• RNA-seq: For unbiased discovery of all bound RNAs

• m6A-seq: Combine with m6A-specific antibodies to correlate binding with modification

Validation Approaches:

• MeRIP assays: To confirm changes in m6A levels of target transcripts

• RNA stability assays: Assess impact on mRNA half-life

• Luciferase reporter assays: Validate binding to specific 3' UTRs

Studies have successfully employed ALKBH5 antibodies in RIP experiments to identify key targets like CK2α in bladder cancer, demonstrating that ALKBH5 reduces m6A modification of CK2α mRNA, thereby affecting its stability .

What experimental design is optimal for investigating ALKBH5's role in stem cell maintenance?

Based on research showing ALKBH5's importance in hematopoietic stem cell (HSC) self-renewal , an optimal experimental design would include:

In Vitro Components:

• HSC isolation and purification protocols

• Colony formation assays to assess self-renewal

• Differentiation assays to evaluate multipotency

• RNA-seq and m6A-seq to identify regulatory targets

• Mechanistic validation of key targets (e.g., Cebpa)

In Vivo Components:

• Conditional knockout mouse models (Alkbh5ᶠˡ/ᶠˡ with Mx1-Cre)

• Competitive bone marrow transplantation assays

• Serial transplantation to assess long-term self-renewal

• Lineage tracing experiments

• Stress recovery models (5-FU challenge)

Key Measurements:

• HSC quantification (LT-HSCs, ST-HSCs, MPPs)

• Cell cycle analysis

• Differentiation potential across lineages

• m6A levels in target transcripts

• Expression levels of stemness genes

Controls and Validations:

• Incomplete vs. complete Alkbh5 knockout comparisons

• Rescue experiments with wild-type Alkbh5

• Comparison with other m6A regulatory enzymes

Research has shown that Alkbh5 deletion compromises long-term self-renewal capacity of HSCs while increasing progenitor cell populations, possibly through regulation of Cebpa m6A modification .

How can I design experiments to evaluate ALKBH5's impact on cancer drug sensitivity and immune checkpoint inhibition?

Building on findings that ALKBH5 affects cisplatin sensitivity in bladder cancer and immune checkpoint inhibitor response in hepatocellular carcinoma :

Drug Sensitivity Assessment:

• IC₅₀ determination with ALKBH5 overexpression/knockdown

• Apoptosis assays (flow cytometry with Annexin V/PI)

• Cell viability time-course experiments

• Combination therapy evaluation

• In vivo xenograft models with drug treatment

Immune Checkpoint Modulation:

• PD-L1, PD-1, and TIM3 expression analysis

• Co-culture systems with immune cells

• Immune cell infiltration assessment

• Cytokine profiling

• Mouse models with intact immune systems

Mechanistic Investigation:

• m6A-seq to identify differentially modified transcripts

• MeRIP validation of key targets

• RNA stability assays for immune-related transcripts

• 3' UTR luciferase reporter assays

• Rescue experiments with wild-type vs. catalytically dead ALKBH5

Translational Evaluation:

• Patient-derived xenografts

• Ex vivo drug sensitivity testing

• Correlation with clinical response data

• Development of biomarker panels

Research has demonstrated that ALKBH5 knockdown decreased cisplatin-induced apoptosis in bladder cancer cells, and ALKBH5 can influence immune checkpoint inhibitor response by modulating TIM3 expression in hepatocellular carcinoma .

What methodologies are effective for studying ALKBH5's role in tumor immune microenvironment?

Based on findings that ALKBH5 influences immune responses in colorectal cancer and hepatocellular carcinoma :

Immune Cell Characterization:

• Flow cytometry panels for tumor-infiltrating lymphocytes

• Single-cell RNA-seq of tumor microenvironment

• Spatial transcriptomics to map immune cell locations

• Cytokine/chemokine profiling

• T cell functionality assays (proliferation, cytotoxicity)

In Vivo Models:

• Syngeneic mouse models with intact immune systems

• ALKBH5 conditional knockout in specific immune cell populations

• Humanized mouse models for human-specific interactions

• Combination with immune checkpoint inhibitors

Molecular Mechanism Analysis:

• m6A profiling of immune-related transcripts

• Analysis of RNA stability for cytokines and checkpoint molecules

• Identification of ALKBH5-regulated immune pathways

• Evaluation of ALKBH5's impact on antigen presentation

Translational Applications:

• Correlation of ALKBH5 expression with immune infiltration in patient samples

• Development of predictive biomarkers for immunotherapy response

• Potential for targeting ALKBH5 to enhance immunotherapy

Studies have shown that ALKBH5 can drive immune suppression in colorectal cancer via the AXIN2-Wnt-DKK1 axis, and impacts the tumor immune microenvironment and response to immune checkpoint inhibitors in hepatocellular carcinoma by targeting TIM3 .

How can ALKBH5 antibodies be integrated with advanced imaging techniques to study subcellular localization and dynamics?

For investigating the spatial and temporal aspects of ALKBH5 function:

Advanced Imaging Approaches:

• Super-resolution microscopy (STORM, PALM) for nanoscale localization

• Live-cell imaging for dynamic translocation studies

• FRET/FLIM for protein-protein interaction analysis

• Correlative light and electron microscopy (CLEM) for ultrastructural context

• Proximity ligation assay (PLA) for detecting protein interactions in situ

Sample Preparation Optimization:

• Fixation methods preserving nuclear architecture

• Permeabilization protocols maintaining RNA-protein interactions

• Multi-antibody labeling strategies for co-localization studies

• Specific organelle markers for precise subcellular mapping

Quantitative Analysis Methods:

• Colocalization coefficients with RNA granule markers

• Nuclear-cytoplasmic distribution ratios

• Signal intensity correlation with m6A levels

• Tracking movement between compartments

Functional Correlation:

• Correlation of localization with cell cycle phases

• Stress-induced redistribution analysis

• Relationship between localization and enzymatic activity

• Changes in cancer vs. normal cells

ALKBH5 has been observed primarily in the nucleus but can show differential localization patterns depending on cellular context and experimental conditions .