Team:Freiburg/Labjournals/SurChem/May

""

<< Back

Labjournal Surchem May

Experiment 12: Optimisation of blocking and first use of PDMS spotting templates

2015.05.28

considerations

  • Try new washing and blocking step. Also use PDMS spotting mask for the first time.

Experiment/Protocol

  • Made 8 APTES/PDITC slides using standard APTES + PDITC surface
  • Use of 4 GOPTS slides from 26.5.2015 to test binding with reduced reaction agressivity of stored GOPTS-slides
  • Spotted 4 GOPTS and 4 APTES/PDITC slides as in Spotting+Blocking
    • one GOPTS and APTES/PDITC slide each was spotted using the PDMS spotting help
    • one GOPTS and APTES/PDITC slide each was incubated over night with GFP

Results

Mean GFP fluorescence intensity of different concentrated spots on GOPTS and APTES/PDITC surfaces
  • Fluorescence intensity on PDITC highest at 500 µg/ml GFP
  • Fluorescence intensity on GOPTS highest at 1680 µg/ml GFP
  • Fluorescence intensity for overnight incubation slightly higher
Mean GFP fluorescence intensity of different concentrated spots on GOPTS surfaces with shutterspeed of 1s and normalised shutterspeed of 4s
  • Intensity values should not differ but they do→ further investigate
Influence of PDMS spotting help on mean GFP fluorescence spot intensity on GOPTS and APTES/PDITC surfaces
  • PDMS spotting help useful on PDITC

Experiment 11: First fluorescence intensity/shutterspeed linearity test

2015.05.26

  • spotted a drop of undiluted GFP on a normal glass slide
  • measured fluorescence intensity with fluorescence microscope with varying shutterspeed

Results

Fluorescence Intensities measured with varying shutterspeed


Experiment 10: GOPTS slides for storage

2015.05.26

considerations

GOPTS-surface may be to agressive for GFP → storage of activated slides may reduce agressivity and allow binding of active GFP

Experiment/Protocol

  • Made 8 slides using standard GOPTS-Protocol
  • stored slides in slideholder at 4°C

Experiment 9: GFP on activated slides (VWR Slides)

2015.05.25

considerations

Because diamond writer is still defect and using scissors isn't that efficient slides weren't labeled this time. Processing took place in different petri dishes taht were labeled on the top to avoid confusion.

The same conditions as in Experiment 8 were used, but slides from VWR were activated instead of the ROTH slides from AG Roth.

Experiment / Protocol

activation
  • 5 slides were cleaned with acetone, isoprop and water
    • 4 of these slides were activated (→ protocol, 2min, 80 L/h)
    • 1 slide was stored for later use as control
  • of the 4 activated slides:
    • 2 were further processed using GOPTS-Protocol
      • 80 µL GOPTS-Solution, sandwich, 1h, RT
      • incubate 30 sec in acetone
      • wash in acetone
      • blow dry and store at 4°C
    • 2 were coated with APTES + PDITC surface
      • APTES-solution, 30 min, RT
      • 2 x 5 min Acetone
      • blow dry
      • 45 min, 110°C (15 min too long → 60 min)
      • 2h in PDITC solution

Use the mixing vessels provided by AG Roth for APTES- and PDITC-solution!
Afterwards wash with the respective solvent (Acetone in case of APTES and DMF in case of PDITC)

spotting

Spotting according to the following pattern:

Spotting model. green: GFP 500 µg/mL, yellow: GFP 1000 µg/mL, red: GFP 1680 µg/mL, violet: Strep-Cy5 25 µg/mL, blue: BSA 10 mg/mL

Spotted slides were incubated for 1h at RT in dark environment and humid conditions (wet towels).

Detection

Remarks

Use 4 slides per condition next time to have at minimum triplicates for evaluation!

Experiment 8: GFP on activated slides (Roth Slides)

2015.05.24

considerations

Use of short names because diamond writer was defect. Slide naming was changed to increasing indices per operator to avoid the need to look up the numbering every time.

# official name
I SN_G_00_001
II SN_G_00_002
III SN_A_00_003
IV SN_A_00_004
V SN_P_00_005
VI SN_P_00_006
horizontal line blank / binding control

Experiment / Protocol

Slide activation
  • All slides were activated according to protocol
    • Flow: 80 L/h
    • Time: 2 min
  • I and II were activated using the standard GOPTS-Protocol
  • III and IV: Standard APTES + PDITC activation
  • V and VI were first activated, incubated in petri dish and activated again right before spotting
Spotting
  • on each slide from left (side with slide name) to right the following three spot columns were spottet:
    • 1:200 GFP 1,58 mg/mL
    • 1:100
    • 1:50
    • 1:10 (far right, spottet as two sport vertical and one right from the highest)
  • slides were incuzbated in humid condition for 1 h
  • spots were dried on air
  • washed with PBS
  • blown dry
Detection
slide name remark
I 11 & 12 faintly visible, 10 nearly gone
II 10 - 12 faintly visible
III 10 - 12 clearly visible
IV 10 - 12 visible but smeared
9 faintly visible
8 visible as barrier for higher conentrations smearings
X 10 - 12 clearly visible
7 very faintly

Experiment 7: NTA-slides on GOPTS - optimization II

2015.05.15 part 1

Entry Slide Number Surface AB-NTA solution NTA incubation Blocking Washing His solution His incubation Stamping
1NW-G-02-007GOPTS460 mM1h-PBS/Imidazole 10 mM1:10/1:1001h1:10
2NW-G-02-008GOPTS460 mM1h-PBS/Imidazole 10 mM1:10/1:10020min1:10
3NW-G-02-009GOPTS460 mM1h-PBS/SDS 0.1%1:10/1:1001h1:10
4NW-G-02-010GOPTS460 mM1h-PBS/SDS 0.1%1:10/1:10020min1:10
5SB-G-02-005GOPTS460 mMovernight-PBS/Imidazole 10 mM1:10/1:1001h1:10
6SB-G-02-006GOPTS460 mMovernight-PBS/Imidazole 10 mM1:10/1:10020min1:10
7SB-G-02-007GOPTS460 mMovernight-PBS/SDS 0.1%1:10/1:1001h1:10
8SB-G-02-008GOPTS460 mMovernight-PBS/SDS 0.1%1:10/1:10020min1:10
9NW-G-02-011-215GOPTS460 mMovernight-PBS/Imidazole 10 mM1:10/1:1001h1:10
10SB-G-02-009-029GOPTS460 mMovernight-PBS/SDS 0.1%1:10/1:10020min1:10

Experimental/Protocol:

Protocols for GOPTS surface, Plasma activation, Ni-NTA on GOPTS surface

  • all slides were washed with acetone, isopropanol and aqua dest.
  • plasma activation: 40-80 L/h, 1min
  • incubation GOPTS: sandwich, 80 µL per sandwich, 1h, RT
  • slides were put in acetone for 30s and dried
  • incubation AB-NTA: sandwich, 80 µL per sandwich, 1h (for entry 1-4)/overnight (for entry 5-10), with humid tissue in petri dish, RT

AB-NTA-solution for 10 slides (c = 460 mM, V = 400 µL)

dd aqua400 µL
NaHCO30.0338 g
AB-NTA0.0479 g
  • slides 1-4 were washed with aqua dest, dried and stored in the fridge overnight

2015.05.16 part 2

Experimental/Protocol:

  • slides 6-10 were washed with aqua dest. and dried
  • all slides were incubated in NiSO4 for 1h

NiSO4 solution (1 %) for 10 slides

NiSO4 * 6 H2O 0.5095 g
aqua dest.50 mL


  • slides were put in washing solutions according to table and immediately dried

PBS/Imidazole (10mM) for 5 slides

Imidazol 0.014 g
PBS 20x 1 mL
aqua dest.19 mL


PBS/SDS (0.1%) for 5 slides

SDS 20% 100 µL
PBS 20x 995 µL
aqua dest.19 mL


  • slides SB-G-02-008, NW-G-02-009 and NW-G-02-010 were additionally washed with aqua dest. afterwards and dried
  • slides were spotted with His-GFP solution (1:10 and 1:100) and StrepCy5 (0.5 µg/mL): spot size 0.5 µL, incubation 20min/1h
  • Slides were dipped in PBS and dried
  • SB-G-02-008 and SB-G-02-009-[029] were stamped on with His-GFP (1:10), then dipped in PBS and dried

Remarks:

  • spots on slides only washed with PBS/SDS immediately „zerfließen“; did not keep spot shape and fused (slide SB-G-02-007)

Experiment 6: NTA-slides on GOPTS and APTES/PDITC - optimization I

2015.05.08 part 1

Entry Slide Number Surface AB-NTA solution NTA incubation Blocking Washing I Washing II His solution His incubation
1SN-G-02-001GOPTS460 mM1h--Tween20/NaCl1:10/1:1001h
2SN-G-02-002GOPTS460 mM1h--Tween20/NaCl1:10/1:1001h
3SN-A-02-001APTES460 mM1hehtanolamineaqua-1:10/1:1001h
4SB-A-02-001APTES460 mM1hethanolamineaqua-1:10/1:1001h
5SB-G-02-001GOPTS460 mMovernightethanolamine-Tween20/NaCl1:10/1:1001h
6SB-G-02-002GOPTS460 mMovernightethanolamine-Tween20/NaCl1:10/1:1001h
7SB-G-02-003GOPTS460 mMovernightBSA/ethanolamine-Tween20/NaCl1:10/1:1001h
8SB-G-02-004GOPTS460 mMovernightBSA/ethanolamine-Tween20/NaCl1:10/1:1001h
9SN-A-02-002APTES460 mMovernightBSA/ethanolamineaqua-1:10/1:1001h
10SB-A-02-002APTES460 mMovernightBSA/ethanolamineaqua-1:10/1:1001h
11NW-G-02-005GOPTS460 mMoveright-PBS-1:10/1:1001h
12NW-G-02-006GOPTS460 mMovernight-PBS-1:10/1:1001h
13NW-A-02-001APTES460 mMovernightethanolaminePBS-1:10/1:1001h
14NW-A-02-002APTES460 mMovernightethanolaminePBS-1:10/1:1001h
15JB-G-02-001GOPTS460 mMovernight--aqua1:10/1:1001h
16JB-G-02_002GOPTS460 mMovernight--aqua1:10/1:1001h
17JB-A-02-005APTES460 mMovernightethanolamine-Tween20/NaCl1:10/1:1001h
18JB-A-02-006APTES460 mMovernightethanolamine-Tween20/NaCl1:10/1:1001h
19SN-G-02-003GOPTS460 mMovernight--Tween20/NaCl1:10/1:100overnight
20SN-G-02_004GOPTS460 mMovernight--Tween20/Nacl1:10/1:100overnight
21SN-A-02-003APTES460 mMovernightethanolamineaqua-1:10/1:100overnight
22SN-A-02-004APTES460 mMovernightethanolamineaqua-1:10/1:100overnight

Experimental/Protocol:

Protocols for APTES + PDITC surface, GOPTS surface, Plasma activation, Ni-NTA on GOPTS surface and Ni-NTA on APTES/PDITC

  • all slides were washed with acetone, isopropanol and aqua dest.
  • activation of GOPTS-slides: 80 L/h, 1 min
  • activation of APTES-slides: 60 L/h, 2 min
  • incubation GOPTS: 1h, sandwich, 80 µL per sandwich, RT
  • incubation APTES: 30 min, RT
  • incubation of APTES-slides in PDITC: in slideholder, RT, 3h

AB-NTA-solution for 18 slides (c = 460 mM, V = 750 µL)

dd aqua750 µL
NaHCO30.063 g
AB-NTA0.09 g
  • slides 5-22 were sandwiched with 80 µL AB-NTA solution overnight with humid tissue in petri dish

2015.05.09 part 2

Experimental/Protocol:

AB-NTA-solution for 4 slides (c = 460 mM, V = 250 µL)

dd aqua250 µL
NaHCO30.021 g
AB-NTA0.03 g
  • incubation slides 1-4 in AB-NTA: 1h, sandwich, 80 µL per sandwich, humid tissue in petri dish
  • slides 5-22 were washed with aqua dest. and dried
  • slides were blocked in ethanolamine or ethanolamine/BSA for 30 min (see table)

Ethanolamin solution for 5 slides (10 %)

Ethanolamine 2 mL
aqua dest.18 mL

Ethanolamin/BSA solution for 5 slides (10 %)

Ethanolamine 2 mL
BSA-solution 10 mg/mL18 mL
  • slides were washed with aqua dest. and dried
  • slides were incubated in NiSO4 solution (1%) for 1h at RT

NiSO4 solution (1 %)

NiSO4 * 6 H2O 1 g
aqua dest.100 mL


Experiment 5: NTA-slides on GOPTS and APTES/PDITC I

2015.05.02

JB-A-02-001
JB-A-02-002
JB-A-02-003
JB-A-02-004

NW-G-02-001
NW-G-02-002
NW-G-02-003
NW-G-02-004

Experimental/Protocol:

Protocols for APTES + PDITC surface, GOPTS surface, Plasma activation, Ni-NTA on GOPTS surface and Ni-NTA on APTES/PDITC

  • APTES slides were activated in plasma generator for 2 min with gas 60-80 L/h
  • APTES solution was prepares according to protocol for 5 slides (with aqua dest. not dd aqua)
  • PDITC solution was prepared according to protocol for 5 slides
  • APTES/PDITC slides were dried in dessicator for approx. 70 min
  • GOPTS slides were washed with acetone, isopropanol and aqua dest.

AB-NTA-solution for 4 slides (c = 460 mM, V = 250 µL)

dd aqua250 µL
NaHCO30.05 g
AB-NTA0.03 g

AB-NTA-solution for 4 slides (c = 2 mM, V = 1000 µL)

dd aqua995.6 µL
NaHCO30.2 g
AB-NTA solution 460 mM4.4 µL
  • slides were sandwiched with AB-NTA solutions and incubated over night
solution slide remarks
460 mM JB-A-02-001had bubbles
JB-A-02-002had bubbles
NW-G-02-001
NW-G-02-002
2 mM JB-A-02-003had bubbles
JB-A-02-004had bubbles
NW-G-02-003
NW-G-02-004

2015.05.03

Experimental/Protocol:

  • slides were washed with water and dried
  • slides were blocked in ethanolamine solution in slideholder for 30 min at RT

Ethanolamin solution for 5 slides (10 %)

Ethanolamine 2 mL
aqua dest.18 mL
  • slides were washed with aqua dest. and dried
  • incubated in NiSO4 solution for 1h (APTES slides) and 2h (GOPTS slides) at RT

NiSO4 solution (1 %) for 5 slides

NiSO4 * 6 H2O 0.2 g
aqua dest.20 mL


  • slides were washed with aqua dest. and dried
  • GOPTS-slides were cleaned for 5-10min in the following solution:

Acetic acid 0.2M/NaCl 0.2M/Tween20 0.1% solution for 5 slides/20 mL

Acetic acid 100% 0.229 mL
NaCl 0.234 g
Tween20 20 µL
aqua dest.20 mL


  • GOPTS slides were washed with aqua dest. and dried
  • all slides were spotted with 7 spots His-GFP (spot size: 0.5 µL, 3 spots 1:100, 4 spots 1:1000) and 1 control spot of StrepCy5 (0.5 µg/mL, spot size 0.5 µL)

His-GFP spot solution 1:100 for 8 slides (=24 spots)

His-GFP stock solution 1.86 mg/mL = 6.43 mM
aqua dest.

His-GFP spot solution 1:1000 for 8 slides (=32 spots)

His-GFP spot solution 1:100
aqua dest.


  • incubation His-GFP: 1h
  • incubation StrepCy5: 30 min for APTES and 45 min for GOPTS
  • spots were let dry out
  • slides were then dipped quickly into aqua dest. (in glass vase) and dried
  • slides were examined under fluorescence microscope and in Microarray Scanner

Remarks:

  • put 1 mL less of NiSO4 solution in slideholders (20 mL is too much)

2015.05.01 SpyTag/Catcher

  • plated SpyTag/SpyCatcher constructs from registry on LB-clm-plates and incubated o/n at 37°C