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Competitive dot blot protocol

Last edited Mon 28 Sep 2020

Stage 1 - Procedure

Materials required

Reagents

  • RNA samples or oligos
  • RNase-free water
  • Wash buffer, eg TBST (1x TBS, 0.1% Tween-20)
  • Blocking Solution
  • Primary antibody
  • Secondary antibody (goat anti-rabbit IgG-HRP, ab97051, or goat anti-mouse IgG-HRP)
  • ECL substrate
  • Nucleoside competitors
  • Streptavidin-AlexaFluor488
  • Methylene blue staining buffer (0.2% Methylene blue in 0.4M sodium acetate and 0.4M acetic acid)

Equipment 

  • RNase-free tubes
  • Heat block
  • Positively charged nylon membrane
  • 10 cm plastic petri dish
  • SG Linker (with 254 nM bulb)
  • Imaging system

Steps

1

Dilute the RNA samples or oligos containing the modification of interest to an appropriate concentration with RNase free water.

2

Denature the diluted RNA sample at 95 degrees in a heat block to disrupt secondary structures for 3 min.

3

Chill the tubes on ice immediately after denaturation to prevent the re-formation of secondary structures of RNA.

4

Cut the positively-charged membrane to an appropriate size.

  • Mark a grid on the membrane lightly with a pencil to guide sample loading.
  • Transfer the membrane to a clean 10 cm plastic petri dish
5

Mix the RNA sample by pipetting up and down and drop 1 µL of RNA onto the positively charged nylon membrane.

Figure 1: Example assay map

6

Transfer the dish with the membrane immediately into the chamber of SG Linker.

  • Remove the dish lid and crosslink the RNA to the membrane (RNA side up) with UV light: 125 mJoule/cm2 at 254 nM.
7

Cut the membrane according to the grid and transfer to a clean 24 well plate.

8

Wash the membrane in 500 μl of wash buffer for 5 min at room temperature with gentle shaking to wash off the unbound RNA.

9

Incubate the membrane (RNA side down) in 300 μl blocking buffer for 1 h at room temperature with gentle shaking.

10

Wash the membrane in wash buffer for 10 min.

11

Dilute the nucleoside competitors serially in blocking buffer to give final concentrations of 1000/200/40/8/0 nM.

  • Incubate each dilution with 1 μg/ml of the antibody you are testing.
12

Incubate the membrane with antibody in 300 μl overnight at 4 degrees C with gentle shaking.

13

Wash the membrane (flip the membrane so the RNA side faces up) three times for 10 min each in 10 ml of wash buffer with shaking.

14

Incubate the membrane (RNA side down) with an appropriate secondary IgG-HRP antibody diluted in blocking buffer for 1 h at room temperature with gentle shaking.

15

Wash the membrane (RNA side up) four times for 10 min each in 10 ml of wash buffer with shaking.

16

Incubate the membrane with ECL substrate (eg ab133406) for 5 min.

17

Expose the membrane with your imaging system and take images on high-resolution mode from 1 s exposure up to 3 min exposure with certain intervals.

18

Wash the membrane in wash buffer for 30 min.

  • Incubate the membrane with Streptavidin-AlexaFluor488 (1:5000 in wash buffer) for 1 h in a black box
  • Exposure the membrane on Alexa 488 with your imaging system.
19

After fluorescence imaging, transfer the membrane to a petri dish containing 10 ml Methylene blue staining buffer.

  • 10 ml Methylene blue staining buffer should be 0.2% Methylene blue in 0.4 M sodium acetate and 0.4 M acetic acid.
  • Incubate the membrane for 30 min with gentle shaking.
20

Wash the membrane with tap water until the background is clean (around 30 s to 60 s wash).

21

Take a photo of the methylene blue stained membrane with your imaging system.