5-Hydroxymethylcytosine (5-hmC) Recombinant Mouse Monoclonal Antibody [PSH06-08]

☑ Relative expression (RE)

Western blot analysis of 5-Hydroxymethylcytosine (5-hmC) on different conjugations with Mouse anti-5-Hydroxymethylcytosine (5-hmC) antibody (HA601351) at 1/1,000 dilution.

Lane 1: Uridine-BSA (negative)
Lane 2: Cytidine-BSA (negative)
Lane 3: 5-Methylcytosine-BSA (negative)
Lane 4: 3-Methylcytosine-BSA (negative)
Lane 5: 5-Hydroxymethylcytidine-BSA (positive)

Lysates/proteins at 2 μg/Lane.

Predicted band size: 66 kDa
Observed band size: 66 kDa

Exposure time: 6 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA601351) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.

Competitive ELISA analysis of 5-hmc was performed by coating wells of a 96-well plate with 50 µl per well of 5-hmc-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 5-hmc monoclonal antibody at concentration of 1 µg/mL with serial diluted 5-hmc and its analogs starting from a concentration of 10,000ng/ml to 0.61ng/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 1/35,000 for one hour at room temperature. Detection was performed using an Ultra TMB Substrate for 8 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 5-hmc and little or no crossreactivity to it analogs.

Immunocytochemistry analysis of HeLa cells labeling 5-Hydroxymethylcytosine (5-hmC) with Mouse anti-5-Hydroxymethylcytosine (5-hmC) antibody (HA601351) at 1/100 dilution.

Cells were fixed in 70% ethyl alcohol for 5 minutes at room temperature, then subjected to acid hydrolysis using 2M HCl in TBST for 30 minutes at room temperature. Permeabilized with 0.1% Triton X-100 in PBS for 15 minutes, and then blocked with 2% BSA for 30 minutes at room temperature. Cells were then incubated with Mouse anti-5-Hydroxymethylcytosine (5-hmC) antibody (HA601351) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488, HA1125) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

beta Tubulin (ET1602-4, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) was used as the secondary antibody at 1/1,000 dilution.

Dot blot analysis of 5-Hydroxymethylcytosine (5-hmC) on different conjugations with Mouse anti-5-Hydroxymethylcytosine (5-hmC) antibody (HA601351) at 1/1,000 dilution. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution for 1 hour at room temperature.

Lane 1: Uridine-BSA (negative)
Lane 2: Cytidine-BSA (negative)
Lane 3: 5-Methylcytosine-BSA (negative)
Lane 4: 3-Methylcytosine-BSA (negative)
Lane 5: 5-Hydroxymethylcytidine-BSA (positive)

Proteins loading: 50ng, 10ng, 2ng, 0.4ng;

Blocking and dilution buffer: 5% NFDM/TBST;

Exposure time: 3 minutes; ECL: K1802.

MeDIP was performed using anti-5-hmC antibody (HA601351) or Normal Mouse IgG. The same amount of unmethylated, 5-Methylcytosine (5-mC) or 5-Hydroxymethylcytosine (5-hmC) DNA standard was spiked in 1ug of genomic DNA isolated from HeLa cells as the control. Realtime PCR was then performed to determine the capture of DNA standard as in % of recovery, which is equivalent to one.