Team:Freiburg/Protocols/Spotting

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Spotting+Blocking

e.g. protocol spotting and Blocking of GOPTS and APTES/PDITC slides

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

  • get GOPTS or APTES/PDITC slides
  • pipette 0.5 µl proteinsloution (e.g. GFP) per spot onto the slide (use following schema)
Spotting model. green: GFP 500 µg/mL, yellow: GFP 1000 µg/mL, red: GFP 1680 µg/mL, violet: 2 µg/ml bBSA, blue: BSA 10 mg/mL
for spotting with PDMS mask:
  • use 5 µl proteinsolution
  • remove proteinsolution after incubation with pipet
  • block for 5 min with 10mg/ml BSA (in PDMS wells)
  • put slide in blocking solution and remove PDMS mask
  1. incubate for 1 h at RT
  2. transfer slides into blocking solution for 30 min-60 min
    • blocking solution for GOPTS: 10mg/ml BSA + 0.5% ethanolamine
    • blocking solution for APTES/PDITC:5mg/ml BSA + 5% ethanolamine
  3. 2x wash slides for 10 min in PBS (shake it)
  4. clean with 1/10 diluted PBS
  5. dry it with nitrogen gun
  6. measure fluorescence at fluorescence microscope
  7. incubate for 45 min in Strep-Cy5 solution (2µg/ml)+ 5-10mg/ml BSA
  8. 2x wash 10 min in PBS (shake it)
  9. clean with H2O form wash bottle and dry it with nitrogen gun
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