In-gel Kinase Assay Protocol Based on Wooten et al,. Sci. STKE, 8 October 2002_Vol. 2002, Issue 153, p.PL15 Paktide Gel: (10%) Monomer solution 833uL 4X Resolving Gel Buffer 624uL * ddH2O 798uL 10% SDS 25uL 10% APS 12.5uL TEMED 4uL * add 25uL Paktide (10mg/mL) after ddH2O addition - Run the gel at 30mA for ~1hour until sufficient separation of molecular weight markers.
- Remove resolving gel and place in 50mL cold SDS-Removal Solution I, agitating gently on an orbital platform shaker. Wash for 1 hour, replacing the solution at least four times, once every 15 minutes.
- Then wash gel for 1 hour with SDS-Removal Solution II with agitation; change the solution twice, once every 30 minutes.
- Denature the proteins by incubating the gel in 50mL cold Denaturation Buffer for 30 minutes with very gentle agitation.
- Replace with 50mL of cold Denaturation Buffer and agitate gently for 1 hour
- Renature the proteins by incubating gel in 50mL cold Renaturation Buffer for 40 minutes with agitation, replacing the Renaturation Buffer at 10-minutes intervals to ensure complete removal of the guanidine hydrochloride
- Place the box containing the gel and 50mL of cold fresh Renaturation Buffer in the cold at 4°C for 3 hours (note: the gel is NOT agitated during this step).
- Replace the solution with 50mL of fresh Renaturation Buffer and incubate the gel for 15 hours (overnight) at 4°C (note: the gel is NOT agitated during this step).
- The next day, wash the gel twice for 30 minutes in 50mL Kinase Assay Buffer at Room Temperature with gentle agitation on the orbital shaker
- Transfer the gel to a seal-a-meal bag and add 7 ml Kinase Assay Buffer containing 100µCi [32P]-g-ATP (20µM cold ATP)
- Seal and shake 4 hr at 30 °C then wash gel with Stop Solution
- Dry gel and analyze by autoradiography
Recipes: 1) SDS-Removal Solution I Final Concentration Stock Amount 50mM Tris-HCl, pH 8.0 1M 12.5mL 20%(v/v) 2-propanol Pure 50mL Add ddH2O to a final volume of 250mL. This volume is adequate for one minigel. 2) SDS-Removal Solution II Final Concentration Stock Amount 50mM Tris-HCl, pH 8.0 1M 12.5mL 1mM DTT Powder 0.039g Add ddH2O to a final volume of 250mL. This volume is adequate for one minigel. 3) Denaturation Buffer Final Concentration Stock Amount 50mM Tris-HCl, pH 8.0 1M 12.5mL 20mM DTT Powder 0.771g 6M Guanidine hydrochloride Crystal 143.3g Add ddH2O to a final volume of 250mL. This volume is adequate for one minigel. 4) Renaturation Buffer Final Concentration Stock Amount 50mM Tris-HCl, pH 8.0 1M 12.5mL 5mM DTT Powder 0.195g 0.04% Tween-20 Pure 0.1mL 100mM NaCl 5M 5mL 5mM MgCl2 100mM 12.5mL Add ddH2O to a final volume of 250mL. This volume is adequate for one minigel. 5) Kinase Assay Buffer Final Concentration Stock Amount 20mM HEPES pH 7.7 1M 5mL 10mM MgCl2 1M 2.5mL 2mM DTT 1M 0.5mL 0.1mM EGTA 1M 25uL Add ddH2O to a final volume of 250mL. This volume is adequate for one minigel. 6) Stop Solution Final concentration Stock Amount | 