Team:Freiburg/Labjournals/SurChem/May
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
- 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
- Intensity values should not differ but they do→ further investigate
- 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
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
Afterwards wash with the respective solvent (Acetone in case of APTES and DMF in case of PDITC)
spotting
Spotting according to the following pattern:
Spotted slides were incubated for 1h at RT in dark environment and humid conditions (wet towels).
Detection
Remarks
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 |
---|---|---|---|---|---|---|---|---|---|
1 | NW-G-02-007 | GOPTS | 460 mM | 1h | - | PBS/Imidazole 10 mM | 1:10/1:100 | 1h | 1:10 |
2 | NW-G-02-008 | GOPTS | 460 mM | 1h | - | PBS/Imidazole 10 mM | 1:10/1:100 | 20min | 1:10 |
3 | NW-G-02-009 | GOPTS | 460 mM | 1h | - | PBS/SDS 0.1% | 1:10/1:100 | 1h | 1:10 |
4 | NW-G-02-010 | GOPTS | 460 mM | 1h | - | PBS/SDS 0.1% | 1:10/1:100 | 20min | 1:10 |
5 | SB-G-02-005 | GOPTS | 460 mM | overnight | - | PBS/Imidazole 10 mM | 1:10/1:100 | 1h | 1:10 |
6 | SB-G-02-006 | GOPTS | 460 mM | overnight | - | PBS/Imidazole 10 mM | 1:10/1:100 | 20min | 1:10 |
7 | SB-G-02-007 | GOPTS | 460 mM | overnight | - | PBS/SDS 0.1% | 1:10/1:100 | 1h | 1:10 |
8 | SB-G-02-008 | GOPTS | 460 mM | overnight | - | PBS/SDS 0.1% | 1:10/1:100 | 20min | 1:10 |
9 | NW-G-02-011-215 | GOPTS | 460 mM | overnight | - | PBS/Imidazole 10 mM | 1:10/1:100 | 1h | 1:10 |
10 | SB-G-02-009-029 | GOPTS | 460 mM | overnight | - | PBS/SDS 0.1% | 1:10/1:100 | 20min | 1:10 |
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 aqua | 400 µL |
NaHCO3 | 0.0338 g |
AB-NTA | 0.0479 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 |
---|---|---|---|---|---|---|---|---|---|
1 | SN-G-02-001 | GOPTS | 460 mM | 1h | - | - | Tween20/NaCl | 1:10/1:100 | 1h |
2 | SN-G-02-002 | GOPTS | 460 mM | 1h | - | - | Tween20/NaCl | 1:10/1:100 | 1h |
3 | SN-A-02-001 | APTES | 460 mM | 1h | ehtanolamine | aqua | - | 1:10/1:100 | 1h |
4 | SB-A-02-001 | APTES | 460 mM | 1h | ethanolamine | aqua | - | 1:10/1:100 | 1h |
5 | SB-G-02-001 | GOPTS | 460 mM | overnight | ethanolamine | - | Tween20/NaCl | 1:10/1:100 | 1h |
6 | SB-G-02-002 | GOPTS | 460 mM | overnight | ethanolamine | - | Tween20/NaCl | 1:10/1:100 | 1h |
7 | SB-G-02-003 | GOPTS | 460 mM | overnight | BSA/ethanolamine | - | Tween20/NaCl | 1:10/1:100 | 1h |
8 | SB-G-02-004 | GOPTS | 460 mM | overnight | BSA/ethanolamine | - | Tween20/NaCl | 1:10/1:100 | 1h |
9 | SN-A-02-002 | APTES | 460 mM | overnight | BSA/ethanolamine | aqua | - | 1:10/1:100 | 1h |
10 | SB-A-02-002 | APTES | 460 mM | overnight | BSA/ethanolamine | aqua | - | 1:10/1:100 | 1h |
11 | NW-G-02-005 | GOPTS | 460 mM | overight | - | PBS | - | 1:10/1:100 | 1h |
12 | NW-G-02-006 | GOPTS | 460 mM | overnight | - | PBS | - | 1:10/1:100 | 1h |
13 | NW-A-02-001 | APTES | 460 mM | overnight | ethanolamine | PBS | - | 1:10/1:100 | 1h |
14 | NW-A-02-002 | APTES | 460 mM | overnight | ethanolamine | PBS | - | 1:10/1:100 | 1h |
15 | JB-G-02-001 | GOPTS | 460 mM | overnight | - | - | aqua | 1:10/1:100 | 1h |
16 | JB-G-02_002 | GOPTS | 460 mM | overnight | - | - | aqua | 1:10/1:100 | 1h |
17 | JB-A-02-005 | APTES | 460 mM | overnight | ethanolamine | - | Tween20/NaCl | 1:10/1:100 | 1h |
18 | JB-A-02-006 | APTES | 460 mM | overnight | ethanolamine | - | Tween20/NaCl | 1:10/1:100 | 1h |
19 | SN-G-02-003 | GOPTS | 460 mM | overnight | - | - | Tween20/NaCl | 1:10/1:100 | overnight |
20 | SN-G-02_004 | GOPTS | 460 mM | overnight | - | - | Tween20/Nacl | 1:10/1:100 | overnight |
21 | SN-A-02-003 | APTES | 460 mM | overnight | ethanolamine | aqua | - | 1:10/1:100 | overnight |
22 | SN-A-02-004 | APTES | 460 mM | overnight | ethanolamine | aqua | - | 1:10/1:100 | overnight |
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 aqua | 750 µL |
NaHCO3 | 0.063 g |
AB-NTA | 0.09 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 aqua | 250 µL |
NaHCO3 | 0.021 g |
AB-NTA | 0.03 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/mL | 18 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 aqua | 250 µL |
NaHCO3 | 0.05 g |
AB-NTA | 0.03 g |
AB-NTA-solution for 4 slides (c = 2 mM, V = 1000 µL)
dd aqua | 995.6 µL |
NaHCO3 | 0.2 g |
AB-NTA solution 460 mM | 4.4 µL |
- slides were sandwiched with AB-NTA solutions and incubated over night
solution | slide | remarks |
---|---|---|
460 mM | JB-A-02-001 | had bubbles |
JB-A-02-002 | had bubbles | |
NW-G-02-001 | ||
NW-G-02-002 | ||
2 mM | JB-A-02-003 | had bubbles |
JB-A-02-004 | had 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