Filter Binding Assay
- In vitro transcribe (body labeled) RNA to test for binding. End-labeled should also work.
- Gel purify transcript and determine specific activity.
- Prepare buffers fresh.
5X Filter Buffer Stock per 100 mL 1X PBS pH 7.6 10X 10 ml 10.5mM MgCl2 1M 1.060 ml 2.5mM DTT 1M 250 μl 0.5mM EDTA 0.5M 100 μl 30% glycerol 100% 30 ml DEPCH20 58.6 ml 5x Binding Buffer Stock per 20 mL 50 μg/ml yeast RNA 10 μg/ml 100 μl 250 μg/ml BSA 25 μg/ml 200 μl filter buffer 5X 20 ml
- Soak top (Protran 0.45 μm nitrocellulose by Whatman) and bottom (Nitran SPC supercharged 0.45 μm nylon by Whatman) membranes in 1X filter buffer at room temp 2 hrs or more prior to use.
Dilute target RNA in 1X binding buffer:
- 0.5 fmol per 15 μl reaction: add the RNA in a 10 μl quantity to 5 μl protein as follows:
- for every six protein concentrations used, need triplicate 15 μl reactions plus a triplicate
no protein control = 21 rxns. Dilute enough for 24 rxns: 12 fmol RNA in 240 μl 1X binding buffer:
- 48 μl 5X binding buffer
- x μl body-labeled RNA (add third)
- (192 - x) μl H2O
- Dilute protein in 1X binding buffer with the initial concentrations of 30, 75, 150, 300, 750, and 3000 nM, so that in the reaction the concentrations will be 10, 25, 50, 100, 250, and 1000 nM. This will likely change after the initial experiment to determine what the optimal range will be. You will need just over 15 μl of each concentration to do the triplicate reactions.
- Aliquot 10 μl RNA/tube. Add 5 μl protein at the designated concentration to each tube, or 5 μl of 1X binding buffer for the negative control.
- Incubate on the benchtop for 30 minutes.
- During incubation, set up the filter apparatus
- Add 85 μl of cold 1X binding buffer to each reaction.
- Rinse wells to be used on apparatus with 100 μl cold 1X binding buffer by pulling buffer through filter with vacuum.
- Add reactions to their proper wells, and filter-bind with vacuum.
- Wash 2X with 400 μl cold 1X binding buffer by pulling vacuum.
- Remove both membranes and expose to phosphorimager screen approx 1 hr, then scan.
- Top membrane = bound RNA
- Bottom membrane = unbound RNA
- Immediately after filter binding experiment, clean and decontaminate equipment.
- This will also be performed using the protein p22 as a negative control.
- Analyze using GraphPad Prism 5 software.
Download Protocol: Filter Binding Assay
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