EMSA PROTOCOL DAY 1: MAKE THE PROTEIN MATERIALS: - TNT coupled reticulocyte lysate KIT (Promega, Cat. #L4610) - Bio-Rad Control 89694; Pre-stained SDS-PAGE std.; Low range, Cat. # 161-0305. - steptavidin alkaline phosphatase (Promega; Cat. # V5591) - Western Blue Stabilized substrate for alkaline phosphatase (Promega, Cat. #S3841) - 1X TBS (20mM Tris, pH 7.5 and 150mM NaCl) - 2.42 g Tris / L - 8.77 g NaCl / L - pH to 7.5 - Promega Transcend tRNA (not provided in TNT kit) (Cat. # L506A) 30L - Transcend Colorimetric translation detection system (Promega; Cat. #L5070) - Strep. A (Streptavidin alkaline phosphatase (Promega; Cat. #V559C) DAY 1: METHOD: 1. Clean everything (i.e. bench, pipettes, etc., …) with RNase-Away. 2. Follow TNT coupled reticulocyte lysate Quick Protocol. Can double volume of tube to have a final of 100 L. 3. Remove TNT kit from –800C. 4. Immediately place TNT RNA Polymerase on ice. 5. Rapidly thaw TNT reticulocyte lysate by hand and place on ice. 6. Thaw all other components at RT then place on ice. 7. Translation procedure: CAN DOUBLE REACTION MIXTURE IN TUBE - Prepare PCR tubes for sample as well as for luciferase control. Components Standard reaction using Transcend tRNA TNT rabbit reticulocyte lysate TNT Reaction buffer TNT RNA polymerase (T7) Amino acid mixture (minus Leucine) 1 mM Amino acid mixture (minus Methionine) 1 mM RNasin ribonuclease inhibitor (40u/L) DNA template (1 g/L)* Transcend biotin-lysyl-tRNA** Nuclease-free water* ** Final volume 25 L 2 L 1 L 0.5 L * can use 2 g of DNA template *** can substitute with DEAE water 8. 0.5 L 1 L 1 L 1 L 17 L 50 L ** do not thaw > 5 times Incubate reaction @ 300C for 90 minutes. (Do in PCR machine). SDS-PAGE GEL: 9. Make 10% SDS-PAGE gel. 10. Aliquot 10 L sample in eppendorf tube and add 5 L of 3X sample buffer. 11. Store remaining sample @ -200C. 12. Place in boiling water bath for 1 minute. 13. Gently spin. 14. Load 10% SDS-PAGE gel. Use 5 L standard (Bio-Rad Control 89694; Pre-stained SDS-PAGE std.; Low range, Cat. # 161-0305). Load luciferase control nearest to standard and sample furthest away. 15. Follow normal protocol for running SDS-PAGE gels. 16. Follow normal protocol for transferring to nylon membrane. 17. Briefly wet the membrane in 1X TBS. - KEEP PROTEIN-CONTAINING SIDE UP! 18. Add 1X TBS with 0.5% Tween-20 and incubate @ RT for 60 minutes. STREPTAVIDIN-AP BINDING & COLOUR DEVELOPMENT: 19. Dilute 6 L streptavidin-AP into 15mL fresh 1X TBS + 0.5% Tween-20. Pour off the 1X TBS with 0.5% Tween-20 (step 18) and add this new solution with streptavidin-AP. Shake gently @ RT for 60 minutes. 20. Pour of the streptavidin-AP solution. Wash 2X for 1 minute each with solution of 1X TBS with 0.5% Tween-20. 21. Wash 2X for 1 minute each with 1X TBS. 22. Add 5mL Western Blue Stabilized substrate for alkaline phosphatase to the membrane for 1 to 15 minutes until the bands of interest are of a desired intensity. - KEEP FOIL COVERED TO PROTECT FROM STRONG LIGHT! 23. Stop reaction by washing membrane for several minutes with deionized water, changing the water at least once. 24. Air dry membrane. Protect from light during prolonged storage. DAY 2: ANNEAL OLIGOS (PROBE) MATERIALS: - H2O bath - 5X Annealing Buffer 125 mM Tris (pH 8.0) 2.5 mM MgCl2 125 mM NaCl MW 121.1 203.3 40.0 mass for 100mL 1.52g 0.06g 0.50g DAY 2: METHOD: Oligos: Want a final concentration of 3.5 pmol/L in the 200 L final volume. Therefore, the initial concentration of oligo is 0.7 nmol. Therefore the FORMULA is = # bases x 330 x 0.7ng = _____ ng For example, for a 20mer with a concentration of 1 g/L, you want 4620 ng, so that would be 4.6 L. 1. In eppendorf tube put the following contents: For 20 base oligo: 5X annealing buffer sense oligo 1 (want 3.5 pmol/L final) anti-sense oligo 2 (want 3.5 pmol/L final) H2O Final volume 2. Place is boiling H2O bath for 5 minutes. 3. Slowly cool to RT (~ 8 hours). 40 L 4.6 L 4.6 L 150.8 L 200 L DAY 3: MAKE PROBE "HOT"& INCUBATE WITH PROTEIN & RUN GEL MATERIALS: - gel apparatus - mixture of 0.5 L of 0.5M EDTA + 39.5 L TE buffer for several tubes. - 5X TBE buffer: 162 g Tris 82.5g Boric acid 60 mL 0.5M EDTA (pH 8.0) / / upto 3 L [from Current Protocols in Molecular Biology (Red Books) … page 2.5A4] 20% Ficoll 400 1.0% sodium dodecyl sulfate 0.25% bromphenol blue 0.25% xylene cyanol (optional; runs ~50% as fast as bromphenol blue and can interfere with visualization of bands of moderate molecular weight, but can be helpful for monitoring long runs). 10X loading buffer - Quick spin columns (G-25 Sepheadex columns (Fine) for radiolabeled DNA purification; Roche; Cat. #1 273 922). - make 2X binding buffers - 1X TBS is gel running buffer NOTE: Swing-bucket rotor better than fixed angle rotor. DAY 3: METHOD: MAKE NATIVE GEL 5X TBE 30% Acrylamide / 0.8% Bisacyrl H2O 14.4 mL 16.8 mL 41 mL 10% APS TEMED 540 L 84 L MAKE PROBES "HOT" 1. In eppendorf tube in HOT LAB add the following: Annealed oligos 5X T4 polynuclotide kinase buffer (Forward Buffer; Gibco) [ - 32P ATP] 10ci/L H2O T4 kinase (Gibco) Final volume 1.5 L (5 pmol) 2 L 2.5 L (Gibco) 3 L 1 L 10 L 2. 370C incubator for 10 minutes. 3. Stop reaction by adding mixture of 0.5 L of 0.5M EDTA + 39.5 L TE buffer. 4. Remove column from storage bag and invert several times to resuspend the medium. 5. Remove top cap from column, then remove bottom tip. 6. Allow column to drain by gravity then discard the buffer. 7. Place column in collection tube, and spin @ 1100 x g (3000rpm in Beckman) for 2 minutes. Discard collection tube and eluted buffer. 8. Keep column in upright position. Into column apply HOT DNA sample to center of column bed. 9. Place column in a second collection tube. 10. Centrifuge for 4 minutes @ 1100 x g (3000rpm in Beckman) for 4 minutes. Save the collection tube as this has the purified HOT DNA. 11. Discard column into HOT waste container. 12. Place L of HOT probe in tube and check radioactivity. Use program #2 (block #2 out and leave the others open). On the print-out, you want the 3rd number (which is 32 P) to be > 1 x 105. 13. Probe can be stored in +40C fridge if necessary. INCUBATE HOT PROBE WITH PROTEIN 14. MAKE LIST OF SEVERAL PROTEIN-OLIGO-BUFFER COMBINATIONS. Then, in several eppendorf tubes add the following: 2X Binding buffer Protein Polydi/dc Hot probe* H2O Final volume 20 L 10 L 2 L 5 L 3 L 40 L * Can decrease the volume of HOT probe used. Start quite HOT (5 L) and decrease if necessary. 15. Incubate @ RT for 30 minutes. 16. Add 10X running dye (4 to 6 L) to each tube. RUN NATIVE GEL 17. Run gel @ 300 Volts for ~ 3.5 hours. Alternatively, run gel @ 220 Volts for ~ 5 hours. Running buffer is 1X TBS. 18. Place gel on filter paper. Cover with plastic wrap and cut paper around the gel leaving ~ 3 cm border of paper all around. Put the gel face-up on gel dryer. Leave NO AIR BUBBLES between the plastic wrap and the gel!!! Dry gel under heat for ~ 4 hours. 19. Put dried gel in cassette and expose film over-night. Alternatively, use phosphorimager plate and expose for ~ 8 hours.