AbFlex® N6-Methyladenosine (m6A) antibody (rAb)

Catalog No: 91261 Format: 100 µg ¥62,000 Buy
Catalog No: 91262 Format: 10 µg ¥17,000 Buy

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Antibody Type:
Recombinant
Isotype:
IgG2a
Purification:
Protein A Chromatography
Host:
Mouse
Reactivity:
Human, Mouse, Not Species Specific

Applications

Immunoprecipitation Validated Dot Blot Validated

Application Notes


Validated Applications:
Dot Blot
Immunoprecipitation (IP):
5 - 10 µg

Immunogen

This antibody was raised against N6-methyladenosine conjugated to BSA.

Buffer

Purified IgG in 140 mM Hepes, pH 7.5, 70 mM NaCl, 32 mM NaOAc, 0.035% sodium azide, 30% glycerol.

 

AbFlex® N6-Methyladenosine (m6A) antibody tested by Dot blot.
3.3 ng of single-stranded DNA oligonucleotides were spotted on to a positively charged nylon membrane and blotted with AbFlex N-6-methyladenosine recombinant antibody (1 µg/ml dilution).
1. unmethylated sequence 1
2. single 6-methyl-adenosine in middle of sequence 1
3. unmethylated sequence 2
4. unmethylated sequence 3
5. single 1-methyl-adenosine in middle of sequence 3

AbFlex® N6-Methyladenosine (m6A) antibody tested by RNA immunoprecipitation.
(A) In vitro RNA transcripts were generated containing either 0 or 10% of the m6A modified base or the A unmodified base and were spiked into 10 µg of total RNA.  IP was performed using 10 µg Abflex N6-Methyladenosine antibody followed by Real Time-qPCR.  (B) IP was performed using 5 µg Abflex N6-Methyladenosine antibody and 30 µg of total RNA (fragmented to 200 bp) from human hematopoetic stem cells followed by Real Time-qPCR on known methylated RNA. Data was provided courtesy of Dr. Alexandra Patmanidi in Prof. Dr. Achim Leutz’s lab, Max-Delbrück-Centrum für Molekulare Medizin (MDC).

Background

AbFlex® antibodies are recombinant antibodies (rAbs) that have been generated using defined DNA sequences to produce highly specific, reproducible antibodies. Each AbFlex antibody contains a 6XHis tag, an avidin tag sequence for enzymatic biotin conjugation using the biotin ligase, BirA, and a sortase recognition motif (LPXTG) to attach a variety of labels directly to the antibody including fluorophores, enzymatic substrates (HRP, AP), peptides, drugs as well as solid supports. AbFlex N6-Methyladenosine (m6A) antibody was expressed as full-length IgG with mouse immunoglobulin heavy and light chains (IgG2a isotype) in mammalian 293 cells.

N6-Methyladenosine (m6A) is an RNA modification on the N-6 position of adenosine. This modification has been found to be abundant in the 3’ UTR and stop codons of mammalian mRNA. m6A is associated with miRNA binding sites suggesting a potential role in epigenetic gene regulation. FTO and ALKBH are demethylases for 6-methyladenosine while a multiprotein complex that includes METTL3 functions as the methyltransferase. Recent findings revealed that m6A is also present on metazoan DNA, suggesting a genuine epigenetic role for this modification in the context of DNA as well.

Bridging Antibody to Improve ChIP, MeDIP and IP

Some isotypes of mouse IgG antibodies do not bind well to protein G-conjugated agarose/magnetic beads, which are commonly used in ChIP, MeDIP and IP experiments. Capture efficiency can be improved by including an anti-mouse IgG bridging antibody because it binds with a strong affinity to both the protein G beads and the mouse IgG antibody. By increasing the affinity of the mouse primary antibody for the protein G bead, the Bridging Antibody for Mouse IgG can help improve the results of all ChIP, MeDIP and IP experiments that use AbFlex® N6-Methyladenosine (m6A) antibody (rAb).

Storage

Some products may be shipped at room temperature. This will not affect their stability or performance. Avoid repeated freeze/thaw cycles by aliquoting items into single-use fractions for storage at -20°C for up to 2 years. Keep all reagents on ice when not in storage.

Guarantee

This product is guaranteed for 12 months from date of receipt.

This product is for research use only and is not for use in diagnostic procedures.

Application Key

  • ChIP = Chromatin Immunoprecipitation;
  • DB = Dot Blot;
  • ELISA = Enzyme-linked Immunosorbent Assay;
  • EMSA = Electrophoretic Mobility Shift Assay
  • FC = Flow Cytometry;
  • ICC = Immunocytochemistry;
  • IF = Immunofluorescence;
  • IHC = Immunohistochemistry;
  • IP = Immunoprecipitation;
  • MeDIP = Methyl-DNA Immunoprecipitation;
  • TR-FRET = Time-Resolved Fluorescence Resonance Energy Transfer;
  • WB = Western Blotting