Try several different lengths of exposure. For this technique, the sample is spotted directly on to the membrane, cross-linked, and then undergoes blotting. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. A dot blot works like a simplified version of a western blot. The m6A RNA Methylation Quantification Kit (Colorimetric) (ab185912) is a complete set of optimized buffers and reagents to colorimetrically quantify methylated N6-methyladenosine (m6A) in RNA. Incubate with ECL reagent for 1 min, cover with Saran wrap (remove the excess solution from the surface), and expose X-ray film in the darkroom. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time.Wash three times with TBS-T (1 x 15 min and 2 x 5 min), then once with TBS (5 min).For optimum antibody dilution, follow the manufacturer's recommendation. Incubate with secondary antibody conjugated with HRP for 30 min at room temperature.Wash three times with TBS-T (3 x 5 min).The hydrophobicity of PVDF makes it an ideal support for binding proteins in electrophoretic and dot blotting applications. Incubate with primary antibody (0.1–10 µg/mL for purified antibody, 1:1,000–1:100,000 for antisera, 1:100–1:10,000 for hybridoma supernatant) in BSA/TBS-T for 30 min at room temperature. PVDF (polyvinylidene difluoride) mem- brane was originally introduced to protein use as an ideal medium for the harsh chemical environ- ment of N-terminal, or Edman degradation, sequencing.Block non-specific sites by soaking in 5% BSA in TBS-T in a 10 cm Petri dish (30 min to 1 h at room temperature).Minimize the area that the solution penetrates (usually 2–4 mm diameter) by applying it slowly. This method is better suited for comparing relative abundance of a target protein in a number of samples in parallel ( low-, medium-, high-throughput ). Using a narrow-mouth pipette tip, spot 2 µL of sample onto the nitrocellulose membrane at the center of the grid.Draw a grid by pencil to indicate the region you are going to blot. Have the nitrocellulose membrane ready.Nitrocellulose membrane (BIO-RAD, Trans-Blot, etc.)
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