METTL16 Antibody

What are the primary research applications for METTL16 antibodies?

METTL16 antibodies are extensively utilized in several key applications within molecular biology and neuroscience research. Western blotting represents the most common application, with recommended dilutions typically ranging from 1:1000 to 1:10000 depending on the specific antibody and experimental conditions . Immunohistochemistry applications generally require dilutions between 1:500 to 1:2000, with optimal results achieved using TE buffer (pH 9.0) for antigen retrieval, though citrate buffer (pH 6.0) may serve as an alternative . For immunoprecipitation experiments, a 1:50 dilution has demonstrated effective results for pulling down endogenous METTL16 protein complexes . Additionally, these antibodies have proven valuable in RNA immunoprecipitation (RIP) assays to investigate direct interactions between METTL16 protein and target RNAs such as MAT2A mRNA .

How can I verify METTL16 antibody specificity in my experimental system?

Verifying antibody specificity is crucial for reliable results. A comprehensive validation approach should include:

• Positive controls: Test the antibody on samples with known METTL16 expression. Validated positive controls include HeLa cells, NCI-H1299 cells, NIH/3T3 cells, and mouse/rat testis tissue .

• Knockdown validation: Perform METTL16 knockdown experiments using shRNA or siRNA approaches. For example, lentiviral vectors (Lenti-sh-METTL16) or AAV-based systems (AAV-sh-METTL16) have demonstrated approximately 60% knockdown efficiency when measured by qRT-PCR and confirmed by western blotting .

• Molecular weight confirmation: Verify detection at the expected molecular weight. METTL16 typically appears at 70-75 kDa on western blots, slightly higher than its calculated weight of 64 kDa (562 amino acids), likely due to post-translational modifications .

• Cross-reactivity assessment: Most commercial METTL16 antibodies show reactivity with human, mouse, and rat samples; confirm specificity across your species of interest .

What tissues and cell lines are recommended as positive controls for METTL16 antibody validation?

For robust validation of METTL16 antibodies, researchers should consider the following positive controls:

Cell lines:

• HeLa cells (human cervical cancer)

• NCI-H1299 cells (human non-small cell lung carcinoma)

• NIH/3T3 cells (mouse fibroblasts)

• MDA-MB-231 and MDA-MB-453 (breast cancer cell lines with confirmed METTL16 overexpression)

Tissue samples:

• Mouse testis tissue (consistently shows high expression)

• Rat testis tissue

• Human colon tissue (for IHC applications)

• Hippocampal tissue (particularly from MWM-trained mice, which show upregulated METTL16)

MCF10A cells (normal breast epithelial cells) can serve as a negative/low expression control in comparative studies with breast cancer cell lines .

How can I determine if METTL16 is directly interacting with a specific target mRNA?

Investigating direct METTL16-RNA interactions requires a multi-faceted approach:

• RNA Immunoprecipitation (RIP): First, confirm the practicality of your anti-METTL16 antibody through western blotting. Then perform RIP by immunoprecipitating METTL16 protein complexes using anti-METTL16 antibody (with IgG as a negative control) and analyze captured RNA by qRT-PCR using primers specific to your target mRNA. For example, researchers successfully demonstrated METTL16 interaction with MAT2A mRNA using this approach, finding significant enrichment compared to IgG controls .

• m6A RNA Immunoprecipitation (MeRIP): To confirm m6A modification of your target mRNA, perform MeRIP using anti-m6A antibodies followed by qRT-PCR for your target transcript. This approach revealed m6A modification of FBXO5 mRNA in breast cancer studies .

• Methylated RNA stability assays: After confirming interaction, assess whether METTL16 affects mRNA stability through m6A modification. Treat cells with actinomycin D to inhibit transcription, then harvest RNA at different time points to determine mRNA half-life in METTL16-knockdown versus control conditions .

• 3'UTR reporter assays: For mapping precise interaction sites, clone the putative binding region (e.g., 3'UTR) into a reporter construct and assess expression in METTL16-knockdown versus control conditions .

What strategies should I employ to investigate METTL16's role in learning and memory formation?

To examine METTL16's function in learning and memory:

How can I optimize METTL16 antibody use for detecting endogenous protein in difficult tissue samples?

For challenging tissue samples:

• Optimized tissue preparation:

  • For neural tissues (e.g., hippocampus), preserve tissue integrity through rapid extraction and flash-freezing

  • Consider perfusion fixation for IHC applications

  • For western blotting, include protease inhibitors and phosphatase inhibitors in lysis buffer

• Enhanced antigen retrieval for IHC:

  • Primary recommendation: TE buffer at pH 9.0

  • Alternative method: Citrate buffer at pH 6.0

  • Extend retrieval time for particularly difficult samples

• Signal amplification strategies:

  • For western blotting: Extend primary antibody incubation to overnight at 4°C

  • For IHC: Consider tyramide signal amplification systems

  • Use specialized detection systems (e.g., SuperSignal West Femto for WB)

• Blocking optimization:

  • Test both BSA and milk-based blocking solutions

  • Consider species-specific protein blocks to reduce background

  • Include longer blocking steps (2+ hours) for high-background samples

What controls are essential when studying METTL16's methyltransferase activity?

When investigating METTL16's m6A methyltransferase function:

How should I design experiments to distinguish between METTL16's direct and indirect effects on target gene expression?

To differentiate direct from indirect effects:

• Direct binding assessment:

  • Perform RIP using anti-METTL16 antibody (1:50 dilution for IP) to identify direct RNA interactions

  • Compare enrichment of putative target RNAs to non-target controls

  • Example: MAT2A mRNA showed significant enrichment in anti-METTL16 immunoprecipitates compared to IgG controls

• m6A modification confirmation:

  • Utilize MeRIP to identify m6A modifications on target transcripts

  • Map modification sites through sequencing

  • Compare modification patterns in METTL16-knockdown vs. control conditions

• Mechanistic dissection:

  • RNA stability assays (actinomycin D chase) to determine if METTL16 affects mRNA half-life

  • Reporter assays with wild-type vs. mutated binding sites

  • m6A reader protein knockdown (e.g., YTHDC1) to determine if effects are m6A-dependent

• Temporal analysis:

  • Time-course experiments following METTL16 manipulation

  • Early changes (0-4h) likely represent direct effects

  • Later changes (12-24h+) may include secondary/indirect effects

How can I address inconsistent METTL16 antibody performance across different experimental conditions?

Troubleshooting recommendations:

• Storage and handling optimization:

  • Store antibody at -20°C in small aliquots to avoid freeze-thaw cycles

  • Confirm antibody stability (typically one year after shipment when properly stored)

  • For 20μl sizes containing 0.1% BSA, avoid additional aliquoting

• Protocol modifications:

  • Adjust antibody concentration based on sample type (try dilution ranges within manufacturer recommendations)

  • For WB: 1:2000-1:10000

  • For IHC: 1:500-1:2000

  • Extend incubation time for weak signals

  • Optimize blocking solutions to reduce background

• Sample preparation refinement:

  • Ensure complete protein denaturation for WB

  • Verify protein extraction efficiency

  • For tissues with high protease activity, increase protease inhibitor concentration

• Cross-validation:

  • Test multiple METTL16 antibodies targeting different epitopes

  • Validate results with complementary techniques (e.g., IF to complement WB findings)

  • Include positive control samples (HeLa cells, testis tissue) alongside experimental samples

What factors should I consider when interpreting conflicting data about METTL16's role in cancer versus neurobiology?

When reconciling seemingly contradictory findings:

• Context-specific regulation:

  • METTL16 appears to play tissue-specific roles: promoting memory formation in neurons while facilitating malignant behavior in breast cancer

  • Consider cellular context when interpreting results

• Target spectrum variation:

  • METTL16 may modify different RNA targets depending on cell type

  • In hippocampal cells, MAT2A is a key target affecting memory formation

  • In breast cancer, FBXO5 stabilization promotes malignancy

• Pathway integration:

  • Examine downstream signaling pathways in each context

  • Consider interaction with other m6A regulatory proteins

  • Assess global m6A changes versus specific target effects

• Technical considerations:

  • Confirm antibody specificity in each tissue type

  • Validate knockdown efficiency across different cell types

  • Consider potential compensatory mechanisms in different tissues

How can I leverage METTL16 antibodies to investigate the relationship between m6A modification and RNA stability?

To explore METTL16's role in RNA stability:

• Combined RIP and stability assays:

  • First identify direct METTL16 targets using RIP with anti-METTL16 antibody

  • Then assess stability of these targets following METTL16 knockdown

  • Example: MAT2A mRNA levels decreased upon METTL16 knockdown in both animal and cellular experiments

• Site-specific mutation analysis:

  • Create constructs with wild-type or mutated METTL16 binding sites

  • Compare stability and expression levels between variants

  • Correlate with m6A levels at specific sites

• m6A reader protein investigation:

  • Examine interaction between m6A readers (like YTHDC1) and METTL16-modified transcripts

  • Determine if stability effects require both METTL16-mediated modification and reader binding

  • YTHDC1 knockdown has been shown to affect response to SAM depletion similar to METTL16 knockdown

• Transcriptome-wide analysis:

  • Combine MeRIP-seq with RNA-seq in METTL16-knockdown versus control conditions

  • Correlate changes in m6A modification with abundance of transcripts

  • Identify common sequence motifs in stabilized/destabilized transcripts

How can METTL16 antibodies be used in studying embryonic development and cellular differentiation?

Recent research has revealed that METTL16 is essential for embryonic development:

• Developmental stage-specific analysis:

  • Use anti-METTL16 antibodies for IHC on embryonic tissues at different stages

  • Track expression pattern changes during development

  • Focus on blastocyst stage, as METTL16 deficiency is embryonically lethal, preventing development beyond the 64-cell blastocyst stage

• Stem cell differentiation studies:

  • Monitor METTL16 levels during stem cell differentiation using western blotting

  • Correlate with global m6A levels and differentiation markers

  • Investigate effects of METTL16 knockdown on lineage commitment

• Transcriptome dysregulation assessment:

  • Compare transcriptome profiles between METTL16-deficient and wild-type embryonic cells

  • Identify key developmental genes regulated by METTL16-mediated m6A modification

  • Focus on pathways essential for early embryonic development

• Methyltransferase activity during development:

  • Compare METTL16 activity between pluripotent and differentiated states

  • Identify developmental stage-specific RNA targets

  • Investigate interaction with developmental signaling pathways

What approaches can identify novel METTL16 targets beyond the established MAT2A and FBXO5 mRNAs?

To discover new METTL16 targets:

• Integrated omics approach:

  • Perform RIP-seq using anti-METTL16 antibody to identify bound RNAs

  • Combine with MeRIP-seq to identify m6A-modified transcripts

  • Compare transcriptomes of METTL16-knockdown versus control cells

  • Look for RNAs that are both bound by METTL16 and show altered stability/abundance after knockdown

• Structural motif analysis:

  • Analyze sequence and structural features of known targets (MAT2A, FBXO5, U6 snRNA)

  • Perform computational screening for similar motifs transcriptome-wide

  • Validate candidates experimentally using RIP and stability assays

• Proximity labeling approaches:

  • Use METTL16 fusion proteins with proximity labeling enzymes

  • Identify RNAs in close proximity to METTL16 in living cells

  • Validate with traditional RIP using anti-METTL16 antibodies

• Cross-linking immunoprecipitation (CLIP):

  • Perform CLIP-seq using anti-METTL16 antibodies

  • Map binding sites at nucleotide resolution

  • Correlate with m6A modification sites and RNA stability