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Latest revision as of 09:05, 10 September 2015

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Glass slide preparation

complete protocol for functionalization of glass slide with APTES/PDITC and Ni-NTA

duration: 2 days

Slide preparation

  • Name the slide
  • Wash with: acetone, isopropanol, aqua dest.
  • Dry with wafergun
  • Activate in plasma generator: gas flow 80 L/h, time 5 min

APTES

  • Prepare APTES-solution: for 5 slides:
    • 18.8 mL acetone
    • 1.0 mL dd aqua
    • 0.2 mL APTES (always close with Parafilm)
  • Incubate slides for 30 min in slideholder filled with APTES-solution
  • Wash 2 x 5 min in slideholder with acetone
  • Dry with wafergun
  • Put slides in new slideholder and bake them in oven at 110 °C for 45 min

PDITC

  • Prepare PDITC-solution: for 5 slides:
    • pyridin 2.0 mL
    • DMF 18.0 mL
    • PDITC 40 mg
  • Cool down slides in slidebox with wafergun (go close with gun)
  • Incubate slides in slideholder for 2 h filled with PDITC-solution at RT
  • Discard PDITC-solution
  • Fill with ethanol and discard ethanol
  • Wash 2 x 5 min in slideholder with ethanol
  • Wash for 5min with acetone in slideholder
  • Discard acetone
  • Dry with wafergun
  • Dry for 15 min in desiccator (Exsiccator)

Ni-NTA

  • 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
  • (use 3 µl of bBSA (200 µg/ml in 1mg/ml BSA) as control spot)
  • 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 (5 µg/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
,