Recombinant FTO Antibody - Anticorpo primario, specific to FTO

Cat. No.: Ab325730

GRADE & PURITY KO Validation

Application

Flow Cytometry, IF/ICC, WB

Host species

Coniglio

Reactive species

consiglio, Umano

Isotype

Rabbit IgG

Conjugation

Unconjugated

Specifications

Product Name

Recombinant FTO Antibody - Anticorpo primario, specific to FTO

Specifiche e purezza

KO Validation

Specie ospite

Coniglio

Specificità

FTO

Immunogeno

A synthesized peptide derived from human FTO

Coniugazione

Unconjugated

Grado

KO Validation

Metodo di purificazione

Protein A purified

Product Line

Prodotti biologici

Stoccaggio e spedizione

Condizioni di conservazione di stoccaggio

Store at -20°C,Avoid repeated freezing and thawing

Spedito in

Ice chest + Ice pads

Stabilità e conservazione

Store at -20°C for one year.

Application Protocols

No item-specific, tested protocols or dilutions are provided for this product. Refer to the CoA/Spec Sheet for any lot-specific guidance.

General starting-point protocols (literature/general guidance; optimize empirically):

• Western Blot (WB):

  • Sample: RIPA or NP-40 lysates with protease inhibitors.
  • Load: 10–40 µg total protein per lane.
  • Blocking: 5% BSA or milk in TBST for 30–60 min.
  • Primary: Start at 1:500–1:2,000 in blocking buffer, 1 h RT or overnight at 4°C.
  • Secondary: Species/isotype-appropriate HRP or fluorescent conjugate, 1:5,000–1:20,000.

• Immunofluorescence/ICC:

  • Fix: 4% PFA (10–15 min) or cold methanol (5–10 min).
  • Permeabilize: 0.1–0.3% Triton X-100 if nuclear FTO is targeted.
  • Block: 1% BSA/3% serum, 30–60 min.
  • Primary: 1:200–1:1,000, 1 h RT or overnight 4°C.
  • Secondary: Fluorophore-conjugated antibody, 1:500–1:1,000; include nuclear counterstain if desired.

• Immunoprecipitation (IP):

  • Use 1–5 µg antibody per 0.5–2 mg lysate; incubate 2–16 h at 4°C.
  • Capture with Protein A/G beads; wash stringently; elute for WB or MS.

These ranges are generic. Confirm compatibility (isotype, species, conjugation) and optimize for your samples.

Biological Roles

• Target overview (literature/general knowledge):

  • FTO (Fat mass and obesity-associated protein) is an Fe(II)/α-ketoglutarate–dependent dioxygenase that catalyzes oxidative demethylation of N6-methyladenosine (m6A) and related methylated nucleosides in RNA. It contributes to dynamic epitranscriptomic regulation affecting RNA processing, stability, and translation.
  • Cellular localization: Predominantly nuclear but can have cytoplasmic pools depending on cell type and conditions (literature).
  • Pathway context: Interacts functionally with RNA methyltransferases (e.g., METTL3/METTL14) and other demethylases (e.g., ALKBH5), impacting gene-expression programs.

• Antibody utility (general):

  • An FTO antibody enables detection, localization, and enrichment of the FTO protein to study RNA methylation pathways and their regulation in diverse biological systems.

• Item-specific biological details (species reactivity, epitope, isotype): Not specified for this item; refer to CoA/Spec Sheet.

Buffer Applications

As an antibody reagent, this product is used within immunoassay buffers rather than serving as a buffer itself.

• Item-specific buffer formulation: Not specified for this item; refer to CoA/Spec Sheet.

• General buffer systems for antibody applications (literature/general guidance):

  • WB/IHC/IF wash buffers: PBS or TBS with 0.05–0.1% Tween-20 (PBST/TBST) to reduce nonspecific interactions.
  • Blocking buffers: 5% BSA or 5% nonfat dry milk in TBST/PBST. Choice depends on assay and secondary antibody.
  • Antigen retrieval (IHC): Citrate buffer pH ~6.0 or Tris-EDTA pH ~9.0, heated (e.g., microwave or pressure cooker) to unmask epitopes—conditions must be optimized for FTO and fixation method.
  • Incubation buffers: PBS/TBS with protein carrier (e.g., 1% BSA) to stabilize the antibody and reduce background.
  • Storage buffer supplements: 10–50% glycerol for −20°C storage; preservatives such as 0.02–0.05% sodium azide for long-term 2–8°C storage if compatible with downstream use (not specified for this item).

Green Alternatives

While green chemistry typically targets organic solvents and reagents, sustainability considerations also apply to antibodies.

• Animal-free, recombinant sourcing (general):

  • Recombinant antibodies avoid ascites production and can reduce animal use, aligning with 3Rs (Replacement, Reduction, Refinement). This item is described as recombinant, which supports these goals.

• Process and waste minimization:

  • Use micro-volume workflows (strip blots, 96-well formats) to reduce reagent consumption and buffer waste.
  • Choose non-azide formulations when possible for downstream biological disposal compatibility (preservative content not specified for this item).

• Packaging and cold-chain:

  • Consolidate orders and plan experiments to minimize repeat shipments and associated cold-chain footprint. This item ships on ice packs; prompt receipt and storage reduce wastage.

• Comparison (general guidance):

  • Ascites-derived antibodies: Higher ethical and biowaste concerns; batch variability can demand more optimization.
  • Recombinant antibodies: Improved consistency; potential to reduce repeat optimization and waste. Specific production host and purification details for this item are not specified.

Pharmaceutical Uses

This product is supplied strictly for research use only; it is not intended for diagnostic, therapeutic, or human/animal use.

• Item-specific pharmacopeial status and excipient role: Not specified for this item; refer to CoA/Spec Sheet.

• General context (literature/general knowledge):

  • Research antibodies may be used in bioprocess and quality control laboratories as analytical reagents to characterize targets in cell lines or process intermediates.
  • They can serve as reference reagents in assay development (e.g., ligand-binding assays) but require independent qualification for GMP environments. Any clinical or manufacturing deployment would necessitate rigorous validation and is outside the scope of this catalog item.

Physical Properties

• Item-specific physical constants (BP, MP, density, pI, extinction coefficient, buffer composition): Not specified for this item; refer to CoA/Spec Sheet.

• Practical notes for recombinant antibodies (literature/general knowledge):

  • State: Aqueous solution or lyophilized solid depending on catalog variant (appearance not specified here).
  • Approximate molecular size: Full-length IgG typically ~150 kDa; Fab ~50 kDa; scFv ~25–30 kDa. Specific format for this item is not specified.
  • Solubility: Highly soluble in physiological buffers (e.g., PBS, Tris-buffered saline) with stabilizers such as BSA, sugars, or glycerol when present (not specified for this item).
  • Stability: Sensitive to repeated freeze–thaw; maintain on ice during use.
  • Storage temperature (from Product Data): Store at −20°C. Avoid repeated freezing and thawing.

• UV/Vis (general): Proteins exhibit A280 absorption; extinction coefficient and A280-to-concentration conversion are sequence-dependent and not specified for this antibody.

Quality and Grades

• Item-specific grade (from Product Data): KO Validation.

  • Meaning of KO (knockout) validation (general definition): Antibody performance is confirmed by loss of target signal in samples where the target gene (FTO) is genetically ablated. This supports specificity to FTO rather than off-targets. Details of the validation dataset (assay type, cell line, controls) are not specified for this item.

• Additional quality attributes (not specified for this item; refer to CoA/Spec Sheet):

  • Isotype/subclass, host species, clonality (mono/polyclonal), recombinant expression system, epitope mapping, immunogen, lot-specific concentration, buffer/format, and preservative content.

• Implications for users:

  • KO validation is valuable for confirming on-target recognition in methods such as WB, IF, IHC, or IP. Nonetheless, assay conditions, species cross-reactivity, and optimal dilutions are experiment-dependent and not provided here.
  • Recombinant production (as stated in the product name) generally offers lot-to-lot consistency versus hybridoma/ascites sources; specific platform details are not provided.

Reaction and Applications

Chemical reaction chemistry is not applicable. This product is an immunoreagent used to detect FTO protein.

• Item-specific tested applications: Not specified for this item; refer to CoA/Spec Sheet.

• General research applications for FTO antibodies (literature/general knowledge):

  • Western blot (WB): Detection of FTO in lysates from cells/tissues; KO controls help verify specificity.
  • Immunofluorescence (IF)/Immunocytochemistry (ICC) and Immunohistochemistry (IHC): Visualization of subcellular/tissue distribution of FTO after fixation; antigen retrieval conditions depend on epitope and fixative.
  • Immunoprecipitation (IP)/Co-IP: Enrichment of FTO for downstream analysis (e.g., MS, activity assays) when the antibody binds native conformations.
  • ELISA: If the antibody is capture/detection-compatible; not guaranteed for this item without validation data.

• Practical tips (general):

  • Use KO or knockdown (siRNA/shRNA) samples as negative controls.
  • Include peptide competition only if the immunogen peptide is available (not specified here).
  • Optimize blocking (e.g., 5% BSA vs. milk) based on Fc/reagent compatibility and background.
  • Validate linear range and loading amounts in WB; titrate antibody concentration empirically.

Reaction Conditions

Classical chemical reaction conditions are not applicable. For immunoassays using this antibody, experimental conditions revolve around binding equilibria and assay optimization rather than stoichiometric reactions.

• Item-specific tested conditions: Not specified for this item; refer to CoA/Spec Sheet.

• General assay-condition guidance (literature/general knowledge):

  • WB: Load 10–40 µg total protein per lane; primary antibody incubation 1–16 h at 4–25°C in blocking buffer; wash with PBST/TBST; HRP- or fluorescent secondary detection.
  • IF/ICC: Fix with 4% PFA (10–15 min) or cold methanol (5–10 min); permeabilize with 0.1–0.5% Triton X-100 if needed; block 30–60 min; primary incubation 1–16 h; image with confocal/epifluorescence.
  • IHC: FFPE sections require heat-induced epitope retrieval (citrate pH ~6 or Tris-EDTA pH ~9); primary incubation 1–16 h; DAB or fluorescence detection.
  • IP: 1–5 µg antibody per 0.5–2 mg lysate; incubate 1–4 h or overnight; capture with Protein A/G resin (isotype-dependent). Isotype and Protein A/G compatibility for this item are not specified.

Safety and Handling

• GHS classification, signal word, H-statements, pictograms: Not specified for this item; refer to the SDS.
• General safety for protein antibodies (literature/general guidance):
  • PPE: Lab coat, gloves, and safety glasses when handling solutions and during spill cleanup.
  • Biosafety: Treat all biological reagents as potentially biohazardous. Use aseptic technique to avoid contamination. Dispose of waste per institutional biosafety procedures.
  • Incompatibilities: Avoid strong oxidizers and proteases. Do not mix with microbial contaminants; maintain sterile technique if sterility is required.
  • Special risks: Repeated freeze–thaw can denature antibodies and generate particulates. Thaw on ice and aliquot to minimize cycles.
  • First aid (general): In case of skin/eye contact, rinse with water. If inhaled (aerosols), move to fresh air. If ingested, rinse mouth. Seek medical attention as needed. Always consult the SDS for authoritative instructions.
• Storage & shipping (from Product Data):
  • Storage: −20°C; avoid repeated freeze–thaw.
  • Shipping: Ice chest + ice pads to maintain cold chain.

Solvent Selection

Not typically applicable to antibodies, which are handled in aqueous buffers rather than organic solvents.

• Item-specific buffer composition and excipients: Not specified for this item; refer to CoA/Spec Sheet.

• General guidance (literature/general knowledge):

  • Preferred media: PBS (pH 7.2–7.6) or Tris-buffered saline with 0.02–0.05% sodium azide (if compatible with application) and optional carrier protein (e.g., 0.1–1% BSA) or 10–50% glycerol for freeze–thaw protection.
  • Avoid: High concentrations of chaotropes, extreme pH (<5 or >9) unless specifically formulated, and organic solvents that denature proteins (e.g., acetone, acetonitrile, methanol) except in fixed-tissue protocols where antibody remains in aqueous phase.
  • Detergents: Tween-20 (0.05–0.1%) in wash buffers can reduce nonspecific binding in immunoassays.

Storage and Reconstitution

• Item-specific storage (from Product Data):

  • Store at −20°C. Avoid repeated freezing and thawing.
  • Shipped in ice chest with ice pads.

• Appearance, concentration, formulation, and reconstitution instructions: Not specified for this item; refer to CoA/Spec Sheet.

• General guidance (literature/general knowledge):

  • Upon receipt: Centrifuge briefly to collect contents. Keep on ice during handling.
  • Aliquoting: Divide into single-use aliquots to prevent freeze–thaw. Use low-protein-binding tubes.
  • Stabilizers: If permitted by your application, include carrier protein (e.g., 0.1–1% BSA) and/or 10–50% glycerol to improve stability at −20°C. Avoid azide when using HRP-conjugated secondaries in peroxidase-based assays or for live-cell applications.
  • Short-term storage: 2–8°C for up to 1–2 weeks may be acceptable for many antibodies if preserved; consult datasheet.
  • Long-term storage: −20°C or below. For multi-year storage, −80°C is often preferred (ensure freeze–thaw is minimized).
  • Thawing: Thaw on ice; mix gently. Do not vortex vigorously.
  • Documentation: Record aliquot map, dates, and freeze–thaw count to maintain reagent integrity.

• Research use note (from Product Data): For research use only.

Structure and Identity

Recombinant monoclonal antibody directed against the FTO protein (Fat mass and obesity-associated protein).

• Item-specific facts (from Product Data):

  • SKU: Ab325730
  • Product name: Recombinant FTO Antibody
  • Grade/Purity: KO Validation
  • Category: Antibody (research use only)
  • Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
  • Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
  • SMILES / InChI / InChIKey / CAS / CID: Not applicable to protein biologics; not specified.

• General structural information (literature/general knowledge for recombinant antibodies):

  • Typical format: IgG with two identical heavy chains (~50 kDa each) and two identical light chains (~25 kDa each) linked by disulfide bonds, forming a Y-shaped heterotetramer (~150 kDa total for full-length IgG). Actual isotype/subclass for this item is not specified.
  • Functional domains: Variable regions (VH/VL) provide antigen-binding specificity to FTO epitopes; constant regions (CH/CL) dictate Fc interactions (isotype not specified).
  • Recombinant production: Sequence-defined expression in a heterologous system to ensure lot-to-lot consistency (platform and host not specified).
  • Antigen: FTO (α-ketoglutarate–dependent dioxygenase; m6A RNA demethylase). Epitope, immunogen sequence, and species reactivity are not specified for this item.

Synthetic Utility

Not applicable. As a recombinant antibody, this product is not used as a chemical building block or reagent in synthetic organic chemistry.

• For experimental planning, see the sections on Reaction & Applications, Buffer Applications, and Application Protocols for guidance relevant to immunoassays.

Target Specificity

• Target: FTO (Fat mass and obesity-associated protein), an Fe(II)/α-ketoglutarate–dependent RNA demethylase.

• Item-specific details not specified for this antibody (refer to CoA/Spec Sheet):

  • Clone ID and clonality
  • Isotype/subclass and host species
  • Epitope/immunogen sequence or domain
  • Species reactivity and cross-reactivity
  • Validated applications and KO validation dataset

• General note: KO validation (from Product Data) indicates specificity was assessed using FTO knockout material; however, assay format(s) and conditions are not provided here.

Domande frequenti

What does KO Validation mean?

KO Validation indicates the product has been validated for use in rigorous antibody validation workflows. Validation may include positive/negative control samples, signal-to-noise testing, and protocol-specific QC. Lot-specific test data is published on the Certificate of Analysis where available.

How should this product be stored?

Store at ?20 °C. Avoid repeated freeze–thaw cycles. Aliquot into single-use volumes before freezing.

How is this product shipped?

This product ships in an insulated container with ice pads. Unpack on arrival and transfer it to the storage condition stated above.

What documentation is provided?

Available product documentation, including Certificates of Analysis (COA), Safety Data Sheets (SDS), and specification sheets, is shown in the product document area. Document availability and access follow the current site policy.