Team:Freiburg/Labjournals/SurChem/June
Labjournal Surchem June
Experiment 22: Comparison between different GFPs (Own,Max,Weber), replicate 1
2015.06.30
GOPTS solution was contaminated!
→ prepared GOPTS aliquot (1mL) from fresh GOPTS stock
fall out in APTES solution → also contaminated ?!
→ prepared GOPTS aliquot (1mL) from fresh GOPTS stock
fall out in APTES solution → also contaminated ?!
Considerations
- prepare slides comparable to slides made in AG Roth
- compare different GFP solutions: GFP used before, new GFP from AG Weber practical, new GFP without tag in cell lysate expressed by ourselves and new GFP with His-Tag expressed by us
- First spotting test with HA-mCHerry-His expressed by us
- use AG-Roth internal DNA binding and spotting control standards
Experiment
- made 4 GOPTS and 4 PDITC slides according to GOPTS surface and APTES + PDITC surface protocol
- measured fluorescence of different GFP-solutions in plate reader
- GFP fluorescence was determined by excitation at 485 nm and measurement at 528 nm
- mCherry fluoroescence: 575 nm → 620 nm
raw data
| solution | first dilution (1/10) [RFU] | second dilution (1/100) [RFU] |
|---|---|---|
| GFP Max' 1 mg/mL | 6086 | 832 |
| Lysate GFP nT | 10311 | 2041 |
| Lysate GFP-2x6His | 7280 | 1013 |
| mCherry | 606 | 224 |
| Weber GFP 8 mg/mL | 5692 | 834 |
| Weber GFP 600 mg/mL | 68461 | 1771 |
blank GFP: 680
blank mCherry: 57
blank corrected
| solution | blank corrected [RFU] |
|---|---|
| GFP Max 1 mg/mL | 5406 |
| Lysate GFP nT | 9631 |
| Lysate GFP-2x6His | 7140 |
| Weber GFP 8 mg/mL | 56240 |
| Weber GFP 600 mg/mL | 683930 |
| mCherry | 549 |
- spotted GFP normalized on GFP Max and mCherry in comparable amount
dilution
| solution | volume [µL] | aqua dest [µL] |
|---|---|---|
| GFP Max 1 mg/mL | 100 | 0 |
| Lysate GFP nT | 56 | 44 |
| Lysate GFP-2x6His | 76 | 24 |
| Weber GFP 8 mg/mL | 10 | 90 |
| Weber GFP 600 mg/mL | 0.8 | 99.2 |
| mCherry | 60 | 40 |
slide usage
| # | spot |
|---|---|
| 1 | Lysate GFP no tag |
| 2 | Lysate GFP-2x6His |
| 3 | Max' GFP-His |
| 4 | Weber GFP 8.6 mg/mL |
| 5 | Weber GFP 600 mg/mL |
| 6 | mCherry |
| 7 | BSA 10 mg/mL |
| 8+9 | bBSA 50 ng/µL |
| 10 | spotting control 0.5 µL |
| 11 | binding control 0.5 µL |
for all spots 2 µL solution were used if not specified otherwise
after GFP fluorescence measurement the bBSA, SC and BC spots were incubated with Strep-Cy5 according to Spotting control Cy5
measurments
- measured GFP fluorescence with microscope
- measured Cy5 fluorescence with microarray scanner
- mCherry was also visible in microarray scanner
Results
Experiment 21: Second GFP dilution experiment on GOPTS and PDITC
2015.06.25
Considerations
- serial delution of GFP should be spotted on GOPTS and APTES/PDITC slides to check how much GFP we need to use
Experiment/Protocol
- 2 GOPTS slides were prepared according to GOPTS-Protocol
- 2 APTES/PDITC slides were prepared according to APTES + PDITC surface
- incubated GFP overnight at 4°C
- used 50µg/ml strep-cy5
- used dilutions:
| Spotnumber | [Protein] |
|---|---|
| 1-2 | 1 mg/mL GFP |
| 3-4 | 0.25 mg/mL GFP |
| 5-6 | 0.0625 mg/mL GFP |
| 7-8 | 0.015625 mg/mL GFP |
| 9-10 | 0.00390625 mg/mL GFP |
| 11 | 50 mM bBSA |
- spotting pattern:
Results
Experiment 20: First GFP dilution experiment on GOPTS
- protocols used :Plasma activation, GOPTS surface
2015.06.19: serial dilution of GFP on GOPTS
Considerations
- serial dilution of GFP should be spotted on GOPTS slides to check how much GFP we need to use
Experiment/Protocol
- serial dilution of GFP was prepared according to the following table and spottet on 2 GOPTS slides with spotting template
| Spotnumber | dilution factor | [GFP] |
|---|---|---|
| 1 | not diluted | 1 |
| 2 | 1:1 | 0.5 mg/mL |
| 3 | 1:2 | 0.25 mg/mL |
| 4 | 1:4 | 0.125 mg/mL |
| 5 | 1:8 | 0.0625 mg/mL |
| 6 | 1:16 | 0.03125 mg/mL |
| 7 | 1:32 | 0.015625 mg/mL |
| 8 | 1:64 | 0.0078125 mg/mL |
| 9 | 1:128 | 0.00390625 mg/mL |
| 10 | 1:256 | 0.001953125 mg/mL |
| 11 | 1:512 | 0.0009765625 mg/mL |
- slides were incubated for 1 h at 4 °C
results
- it was impossible to evaluate the fluorescence measurement, because most of the spots weren't even visible, so probably the surface wasn't good enough or the GFP was already denaturated
Experiment 19: Ni-NTA storage experiment
2015.06.15
Experiment/Protocol
- Made 4 standard APTES/PDITC slides and 2 slides using iRIf-slides using standard APTES + PDITC surface
- Made 4 GOPTS slides on standard glass using standard GOPTS-Protocol
- All 8 slides were sandwiched with AB-NTA solution (0.042g AB-NTA, 0.029g NaHCO3, 350 µl aqua dest.) by adding 80 µl
2015.06.16
considerations
- continue Ni-NTA slides from previous day
Experiment/Protocol
- finished Ni-NTA slides according to Ni-NTA protocol
- every washing/blocking solution was prepared with HBS instead of PBS
- Nickelsulfate was deluted in water to prevent falling out
- Ni-NTA-slides were stored at 4 °C for 2 weeks
Results
- Results mentioned in Experiment 28
Experiment 18: Measure dark current
- protocols used :-
2015.06.13
considerations
- measure dark current to determine its influence on fluorescence measurements
Experiment / Protocol
- measured dark current by taking unlit photos in the fluorescence microscope at differing shutterspeed
Results
- Influence of the dark current on the fluorescence intensity measurements is negligible due to its small magnitude
Experiment 17: Second fluorescence intensity/shutterspeed linearity test
- protocols used :-
2015.06.13
considerations
- test spot intensity/shutterspeed linearity
Experiment / Protocol
- diluted GFP stock solution 1:10 and 1:100
- spotted 1 µl GFP stock solution and dilutions on glass slide
- measured fluorescence intensity of the three spots with fluorescence microscope at different shutterspeeds
Results
- linearity of fluorescence intensity and shutterspeed up to an intensity of ca. 2500 confirmed
Experiment 16: Remeasuring of the GFP stock concentration
- protocols used :-
2015.06.13
considerations
- remeasure GFP stock concentration by using the nanodrop
Experiment / Protocol
- measured GFP stock concentration by using the nanodrop
Results
GFP stock concentration
- mean GFP stock concentration was measured: 1105.5 mg/ml → differs from previous measurements
Experiment 15: Ni-NTA surfaces/Test imidizole washing
2015.06.13
Considerations
- prepare 2 iRIf slides with PDITC-surface for the iRIf group to stamp on
- start again with Ni-NTA surface on GOPTS and PDITC surfaces
Experiment / Protocol
Protocols used: Plasma activation,GOPTS-Protocol, APTES + PDITC surface, Spotting+Blocking, Ni-NTA
- Made 6 standard APTES/PDITC slides and 2 slides using iRIf-slides using standard APTES + PDITC surface
- Made 4 GOPTS slides on standard glass using standard GOPTS-Protocol
- iRIf-slides were stored @ 4°C for further use by the iRIf group
- the remaining 10 slides (6x APTES/PDITC and 4x GOPTS) were treated with Ni-NTA according to Ni-NTA protocol
2015.06.14
Considerations
- continue Ni-NTA slides from previous day
Experiment / Protocol
- finished Ni-NTA slides according to Ni-NTA protocol
- Nickel loading step was first performed in PBS → fall out of salt
- changed to water blocked again for 1 h
- spotted GFP in concentrations of 1600 mg/mL, 1000 mg/mL & 500 mg/mL according to Spotting+Blocking protocol
- GFP was diluted in 1/10 PBS to preserve Ni-NTA-Chelate
- spotted slides were stored o/n @ 4°C
Results
- Nickel-solutions should not be prepared in PBS
- Low solubility constant of 4.74 x 10-32 for Ni3(PO4)2 1)
- Change to HBS instead of PBS in the whole protocol may solve this issue
- Only in the slide not washed with imidazol (APTES1) there is an increase in intensity with increasing GFP-concentration
- In slide APTES4 the retained fluorescence is much higher than in the other slides → one slide wih good surface?
- Replication of this experiment may lead to advanced slide handling and thus to results with higher consistency
- The GOPTS-slides (with or without imidazol wash) retained much less fluorescence than the PDITC-derived slides
2015.06.15
Considerations
- continue with Ni-NTA slides from previous days
- washing steps of GFP loaded slides should be performed with 20 mM Imidazol in PBS or PBS only for 2 GOPTS and 3 PDITC slides respectively
Experiment/Protocol
- His-GFP solution spots were removed with pipette (3 µl) from GOPTS slides. APTES slides were completely dry so blocking solution was immediately added.
- all slides were incubated for 10-15 min with 10 mg/ml BSA solution (3 µl spots) in the wells
- slides „were glided“ into HBS solution pH 7.5 and wells were slowly flooded with HBS before the spotting mask was removed
- Washing 2×10 min HBS pH 7.5 (no shaking)
- Washing 10 min in 1/10 diluted HBS (1 APTES, 2 GOPTS) OR 10 min in 1/10 HBS with 20 mM Imidazol (5 APTES, 2 GOPTS)
- Dried with wafergun and fluorescence was measured
- bBSA ans BSA spots were covered with 5 µl of Strep-Cy5 solution (2 µg/ml)+BSA (10 mg/ml)
- Washing 2×10 min in HBS pH 7.5 (no shaking)
- Washing 10 min in 1/10 diluted HBS (diluted in aqua dest.)
- Slides were dried with wafer gun and Cy5 fluorescence was measured in microarray scanner
Results
- in general there was less unspecific binding on the APTES/PDITC slides, than on the GOPTS slides
- for the GOPTS slides the use of imidazole during the washing step reduced the unspecific binding
- for APTES/PDITC suprisingly the slide that was washed without imidazole had the fewest unspecific binding (StrepCy5)
Experiment 14: Reproducibility test of GOPTS and PDITC slides (replicate experiment 11+12)
2015.06.12
considerations
- repeat GFP-spotting experiment the third time
- create measurable iRIf slides with GFP
Experiment / Protocol
Protocols used: Plasma activation,GOPTS-Protocol, APTES + PDITC surface, Spotting+Blocking
- Made 2 standard APTES/PDITC slides and 2 slides using iRIf-slides using standard APTES + PDITC surface
- Made 4 GOPTS slides on standard glass using standard GOPTS-Protocol
- one GOPTS slide has one spot with reduced volume (2 µL) and one spot less at 1000 µg/mL and 500 µg / mL respectively
- one APTES iRiF slide has different spotting scheme
- used 50 µg/ml bBSA for control spot
Results
Experiment 13: Optimitation of washing steps and preparational process for basic slides (replicate experiment 11)
2015.06.02
considerations
- Try new spotting and blocking protocol.
- Changes from protocol (28.5.2015):
- Changed washing with H2O in step 3 and 5 to PBS
- Measure fluorescence of GFP before Strep-Cy5 inkubation
Experiment/Protocol
- Made 4 APTES/PDITC slides using standard APTES + PDITC surface
- Made 4 GOPTS slides using standard GOPTS-Protocol
- three APTES/PDITC and three GOPTS slides were spotted like described in Spotting+Blocking one of each was an iRiF slide
- one APTES/PDITC and one GOPTS slide were spotted using PDMS spotting masks
Results
- Tendencies from 28.5.2015 experiment confirmed
- Fluorescence under „wet“ conditions (with PBS) much higher
- Intensity values should not differ but they do→ further investigate
- PDMS spotting help useful on PDITC →confirm results from 28.5.2015
