Methylene blue-mediated photocrosslinking reactions are a complement to UV crosslinking reactions. Methylene blue belongs to the phenothiazine group of dyes. Due to its ring structure, methylene blue can bind to nucleic acids at lower concentrations and insert bases. At higher concentrations, methylene blue is cationic and binds to the phosphodiester backbone by electrostatic forces. This experiment is from "RNA Laboratory Guidebook", edited by Xiaofei Zheng.

Operation method

Experimental detection of protein-double-stranded RNA interactions by methylene blue-mediated photocrosslinking reaction

Principle

Methylene blue-mediated photocrosslinking reactions are a complement to UV crosslinking reactions. Methylene blue belongs to the phenothiazine group of dyes. Due to its ring structure, methylene blue can bind to nucleic acids at lower concentrations and insert bases. At higher concentrations, methylene blue is cationic and can bind to the phosphodiester backbone by electrostatic forces.

Materials and Instruments

RNA
Methylene blue aqueous solution Binding buffer RNase storage solution SDS sampling buffer Vitamin C aqueous solution

Move

I. Materials and equipment

1. Methylene blue (MB) aqueous solution, 2.5 mg/ml. -20°C. Store away from light.

2. [ ^α-32P ]-labeled RNA. dsDNA can bind to MB and increase the MB concentration required for the cross-linking reaction if the transcription template is not digested by DNase. If complementary ssRNA is used in the procedure, annealing is required (mix 2 to 5 times the isotopically labeled RNA with the complementary end-labeled RNA, heat for 5 min, and cool slowly at room temperature. Annealing of RNA is usually done before the start of the experiment).

3. 10X binding buffer, the formula is determined according to the actual operation, usually, 1X binding buffer contains 10 mmol/L Tris-HCl (pH 7.5), 20~100 mmol/L KCl, 2 mmol/L MgCl₂, 0.5 mmol/L DTT.

4. RNase stock solution.

RNase A: 10 mg/mL RNase A was dissolved in 10 mmol/L Tris-HCl (pH 7.5), 15 mmol/L NaCl, boiled for 15 min, slowly cooled and stored at -20℃.

RNase T1: 100000 U/ml.

RNase V1: 700 U/ml.

5. 5X SDS Sampling Buffer: 10% SDS, 1 mol/L Tris-HCl (pH 6.8), 50% glycerol, 0.03% bromophenol blue, store at room temperature, add 5% β-mercaptoethanol prior to use.

6. 1 mol/L vitamin C aqueous solution (dissolved in DEPC water), preferably prepared before use.

Operation Methods

1. 10 μl of binding system add 1 μl of 10X binding buffer, 5-10 ng of [ ^α-32P ]-labeled RNA, 100 ng of purified protein or 10 μg of protein crude extract, make up 10 μl of water, and react for 5-10 min at room temperature (the reaction conditions need to be adjusted according to the proteins and nucleic acids to be detected).

2. Place the reaction tube in an ice bath, add an appropriate amount of 2.5 mg/ml MB master batch to a final concentration of 0.1 ng/μl~2 ng/μl, blow and mix well; add an appropriate amount of vitamin C and Tris-HCl (pH 7.8), which is 2 times the number of moles of the added vitamin C, in order to adjust the pH of the reaction solution, blow and mix well.

3. Illuminate for 5~20 min.

4. Prepare 10X RNase Mix: 10 μg/μl RNase A, 3 U/μl RNase T1, 0.35 U/μl RNase V1. Add 1 μl of 10X RNase Mix to each tube of reaction solution and react at 37℃ for 30 min (this step can be omitted if purified proteins are used in the experiment).

5. Add 4 μl of 5X SDS Sampling Buffer and electrophoresis.

6. After electrophoresis, the gel can be stained with Caulophylline blue to see if the samples are consistent or if protein-protein cross-links are present due to excess MB. Afterwards, X-rays are radiographically autoradiographed (dsRNA-protein cross-linking complexes show positive bands in the results of radiographic autoradiography).

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