Team:Freiburg/Protocols/Ni NTA 2

_

""

<< Back

Ni-NTA 2.0

Surface functionalization of GOPTS & PDITC with Ni-chelated NTA (improved protocol)

material: chemicals, used kits, …
duration: … min

Surface preparation

  • prepare 80 µL 457 mM AB-NTA, 1 M NaHCO3 solution per slide sandwich
    • use 0.06 g N,N-Bis(carboxymethyl)-L-Lysine-hydrate and 0.042 g NaHCO3 in 500 µL aqua dest
    • use 0.096 g N,N-Bis(carboxymethyl)-L-Lysine-hydrate and 0.067 g NaHCO3 in 800 µL aqua dest
    • use 0.108 g N,N-Bis(carboxymethyl)-L-Lysine-hydrate and 0.076 g NaHCO3 in 900 µL aqua dest
  • put 80 µL on one slide and put the other one upon → Make sure the upper sides were laid together
  • incubate o/n
  • prepare blocking solutions:
    • blocking solution for GOPTS: 10mg/ml BSA + 0.5% ethanolamine in PBS
    • blocking solution for APTES/PDITC:5mg/ml BSA + 5% ethanolamine in PBS
  • transfer slides into blocking solution for 30 min-60 min
  • 2x wash slides for 10 min in PBS (shake it)
  • prepare 20 mL 1 % (w/v) NiSO4 solution per 5 slides (= 0.2 g/20 mL)
  • incubate slides in slide holder with nickel sulfate for 1 h
  • 2x wash slides for 10 min in PBS (shake it)
  • prepare 1/10 PBS-dilution in aqua dest and check for pH (should be 7-8)
  • wash slides in diluted PBS-solution
  • Dry slides with wafer gun

Spotting and washing

  • spot using 11-spot mask
  • incubate o/n @ 4°C
  • remove protein solution with pipette
  • incubate twice 5 min with 10 mg/mL BSA solution in the wells
  • (flush slide with PBS)transfer slide into PBS solution and remove mask
  • wash 10 min in PBS
  • wash 10 min in PBS (with 20 mM Imidazol, 27.2 mg in 20 mL PBS)
  • wash 10 min in 1/10 diluted PBS
  • dry with wafer gun
  • measure fluorescence
  • incubate for 45 min in Strep-Cy5 solution (2µg/ml)+ 5-10mg/ml BSA
  • 2x wash 10 min in PBS (shake it)
  • wash 10 min in 1/10 diluted PBS
  • dry with wafer gun
  • measure Cy5-fluorescence in microarray scanner
,