WangLab:Purify Protein Invitro Kinase Assay

Measure protein expression with absorbance spectrum:

 * 1) Set up the configuration, including scale, spectrum, 400-650 should be enough for FRET indicators.
 * 2) Clean the asymmetric tube (all crystal).
 * 3) Add blank solution, hit Zero, hit baseline.
 * 4) Add 1ml indicator solution, hit start, save the data file *.dsw in the correct directory.

Purify Protein and In vitro kinase assay:

 * 1) Transform PRSET plasmids to JM109 (DE3) (from Promega).
 * 2) Under ex 480, green gargo, pick up bright colonies.
 * 3) put in 2ml 100uM amp LB medium, o/n shake 250rpm 37oC.
 * 4) take bacteria medium, put in 100ml 100uM amp LB medium, shake 250rpm, 2-4hr, monitor the OD 600 reading, 0.2-0.4, dilute into (+400ml) 500ml 100uM amp LB medium, add 0.4mM IPTG to induce, shake o/n, RT, with air venturation.
 * 5) spin down 7000rpm, 10min.
 * 6) add 15 ml bper+0.5 protease coctail tablet+100uM PMSF, completely suspend gently, gentle rock at RT 10min, covered with foil.
 * 7) spin 15000rpm 15 min. filter through (optional). Add Ni-NTA agarose beads 1ml, gentle rock at RT 1hr.
 * 8) prepare the column, rinse with 2x10ml 1xTNS, 50mM Tris,HClph=7.4, 300mM NaCl.
 * 9) Rinse 1x10ml 50mM Tris,HClph=7.4, 300mM NaCl, 10mM imidazole.
 * 10) elute with 1x10ml 50mM Tris,HClph=7.4, 300mM NaCl, 100mM imidazole.
 * 11) dialysis the protein solution 4oC, 4hr-o/n, in 50 mM Tris pH 8, 100 mM NaCl, 10 mM MgCl2, 2mM DTT. 10x Src kinase buffer stock solution: 500 mM Tris pH 8, 1 M NaCl, 100 mM MgCl2, dilute 10x, add 1:1000 2M DTT.
 * 12) Measure the absorbance following the above protocol.
 * 13) YFP (extinction coefficient) EC=77000 M-1 CM-1, CFP EC=32500 M-1 CM-1, GFP EC=62000 M-1 CM-1. So Concentration=reading/EC

In vitro kinase assay

 * 1) Find the 3-way cuvette. Side line facing the emission, small aligned holes for excitation. Clean the cuvette.
 * 2) add 100 ul of indicator solution
 * 3) put in the holder and cover the cover. Make sure to ajust the aperture to ex=0.5-1 and em=0.5-1
 * 4) Hit F10, select HV#950 ON and HV#440 ON.
 * 5) Define Exp.
 * 6) Emission Scan
 * 7) Data acquisition paramter:
 * 8) *Number of scan:				1
 * 9) *Start:470  End: 530
 * 10) *Incremen: 2nm
 * 11) *Integration time: 1sec
 * 12) *Excitation monochrome: 434
 * 13) *Acquisition mode: s / r
 * 14) Correct factor file: Mcorrect.spt
 * 15) *Measure the peak 526/476, record the number as time goes on, calculate the ratio.
 * 16) *Add Src, Add ATP.
 * Esp, Esp, Run experiment, Go.

For Absorbance:

 * Scan, connect on line.
 * Baseline checked
 * Zero for the blank.
 * Start for the samples.
 * Save data as .csv for ascII format

For Fluorometer:

 * Other functions, output to ascII, F1 to pickup files from directory.

For Platereader:

 * Connect, move in. measurement configuration.
 * Dynamics: 3-5 cycles without ATP.
 * 60 cycles after ATP, 2 min interval. 30 flashes.
 * Increasing step: 5 nm.
 * Band width 5nm. Excitation wavelength: 420nm.
 * Emission: 471 to 531. temperature: 30oC.