m6A Mouse Monoclonal Antibody [A8F2]

Dot blot analysis of N6-methyladenosine (m6A) using HA601049.

Loading: Total RNA extracted from HeLa
Blocking Buffer: 5% NFDM/TBST;
Incubation time and temperature: 1 hours at room temperature ;

Primary Antibody: HA601049
Dilution: 2 ug/ml
Dilution Buffer: Primary Antibody Dilution Buffer form Beyotime;
Incubation Time and Temperature: Overnight at 4 ℃ ;

Secondary Antibody: Goat anti-mouse IgG-HRP antibody (HA1006);
Dilution: 1:100,000
Dilution Buffer: 5% NFDM/TBST;
Incubation Time and Temperature: 30 minutes at room temperature;
Exposure time: 60/120 seconds;

RNA dot blot protocol:
1. Denature of HeLa/HEK293 total RNA at 95℃ for 5 minutes;
2. Load total RNA on nitrocellulose membrane (Pore size: 0.2um, BioTrace NT Nitrocellulose Transfer Membrane from Pall Corporation (P/N 66485));
3. Crosslink RNA on membrane with a UV light(15w) for 3 minute;
Followings are standard Western blot procedures.
4. Block the membrane with 5% milk in TBST at room temperature for 1 hour;
5. Wash the membrane with TBST for 3 times, 5 minutes each;
6. Incubate with primary antibody at 4℃ overnight;
7. Wash the membrane with TBST for 3 times, 5 minutes each;
8. Incubate the membrane with HRP-conjugated secondary antibody in 5% milk at room temperature for 30 minutes;
9. Wash the membrane with TBST for 3 times, 5 minutes each;
10. Incubate the membrane in the ECL substrate for 3 minutes.
11. Develop films in a dark room until get an appropriate signal.

Competitive ELISA analysis of m6A was performed by coating wells of a 96-well plate with 50 µl per well of m6A -BSA diluted in carbonate/bicarbonate buffer, at a concentration of 1 µg/mL overnight at 4℃. Wells of the plate were washed, blocked with 1%BSA blocking buffer, and incubated with 100 µl per well of m6A monoclonal antibody at concentration of 1 µg/mL with m6A and its analogs at a concentration of 10ug/ml for 1 hours at room temperature. The plate was washed and incubated with 50 µl per well of an HRP-conjugated goat anti-mouse IgG secondary antibody at a dilution of 1:10,000 for one hour at room temperature. Detection was performed using an Ultra TMB Substrate for 10 minutes at room temperature in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm.

This antibody demonstrates high specificity for m6A and little or no crossreactivity to it analogs.

MeRIP-qPCR showed that there was m6A modification in the 3-UTR region of SENP1. PCR products were used for gel electrophoresis for data visualization.

PMID: 38822351