The method of immobilizing cDNA library-expressed proteins on nitrocellulose or nylon membranes and then screening them with labeled RNA is the most commonly used technique in screening RNA-binding protein studies. This experiment is from the "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Liquid-phase screening of RNA-binding proteins in cDNA expression libraries
Principle
The method of immobilizing cDNA library-expressed proteins on nitrocellulose or nylon membranes and then screening them with labeled RNA is the most commonly used technique in screening RNA-binding protein studies.
Materials and Instruments
TY Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
Storage solution Lysozyme dry powder Bacterial lysis buffer Glycerol aqueous solution
1. 2X TY: 16 g peptone, 10 g yeast extract, 5 g NaCl, water fixed to 1 L, autoclaved.
2. Stock solution: 1 mol/L IPTG; 1 mol/L DTT; 100 mmol/L PMSF (prepared with anhydrous isopropanol); stored at -20℃.
3. lysozyme dry powder, stored at -20℃.
4. Bacterial lysis buffer: 150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA, 1% NP-40 (or Igepal-630), stored at 4°C. Take 10 ml before use and put it in the lysis buffer. Before use, take 10 ml in an ice bath, add 10 μl of 1 mol/L DTT, 100 μl of 100 mmol/L PMSF and about 20 mg of lysozyme.
5. 40% glycerol in water, autoclaved and stored at 4℃.
Operation method
1. Transform the plasmid cDNA expression library into E. coli, and incubate it in the culture medium containing antibiotics at 37℃ with shaking until the OD600 reaches 0.5~1.0, and store the culture products at 4℃. Before use, take out a portion of the library and dilute it step by step, then smear the plate, count the number of clones and calculate the library titer.
2. Add 2 ml of 2X TY containing antibiotics into the culture tube, inoculate 250 bacterial clones in each tube, and incubate at 37℃ with shaking until the OD600 reaches 0.5.
3. 10 mmol/L IFTG induced expression of lacZ fusion protein and incubate at 37℃ for 3 h or overnight.
4. 1.5 ml of the culture was labeled and collected by centrifugation at 14000 g for 1 min at room temperature, and the remaining 0.5 ml was stored at 4℃.
5. Place the bacteria obtained by centrifugation on ice, resuspend with bacterial lysis buffer and ice bath for 30 min.
6. 14000 g, 4 ℃ centrifugation 15 min, at the same time, take a clean centrifuge tube in the ice bath, labeled with 20 μl of 40% glycerol; centrifuged sample tube is also placed in the ice bath, take 20 μl of the supernatant of the tube and add to the clean centrifuge tube containing glycerol, blowing and mixing, then obtain the bacterial extracts containing the cDNA library proteins.
7. Immediately freeze the bacterial extracts in dry ice/ethanol or liquid nitrogen and store at -70°C. (Thaw in ice bath when ready to use. (Ice bath to dissolve for use, and again flash freeze with dry ice/ethanol or liquid nitrogen before storing at -70°C.)
8. Detect the extracts with a binding site probe (gel-shift or UV crosslinking). If RNA binding activity is present in the collected extracts, the specificity of this RNA binding activity is tested with a negative control probe together with the RNA probe under study.
9. For samples with RNA-binding activity in the extract, find the remaining 0.5 ml of bacterial culture product stored according to the labeling and count the sample titer; inoculate 40 culture tubes with 25 bacterial clones each and incubate at 37°C with shaking until an OD600 of 0.5 is reached.
10. Repeat steps (3)~(9). Once the bacterial protein extract in a particular tube has been shown to have RNA binding activity, plate the corresponding labeled bacterial culture sample to obtain a single clone and inoculate 20 culture tubes with 5 clones of bacteria each; repeat steps (3)~(9); for the final round of screening, inoculate 20 culture tubes with only 1 clone per tube.