1. Prepared coating solution: used Protocol from “Abeam: direct Elisa coating”. Took 0.78g Na2CO3, 1.5g NaHCO3 and dissolve it in 250ml H2O, pH: 9.5.
   
2. Dilute stock solution of GFP (1350µg/ml) in coating buffer for coating concentration of 20µg/ml.
   
3. Coted 93 wells with 50µl coating solution
   
4. Incubated for 2h at room temperature
   
5. Washed the wells twice with PBS (V=200µl)
   
6. Blocked it with 5% nonfat dry milk in PBS (2.5g in 50ml PBS), blocked coated wells with 200µl solution.
   
7. Incubated for 2h at room temperature.
   
8. Washed blocked wells twice with PBS (V=200µl)
   

Measured the emission from top Measured the emission from bottom

Measured the plate from 05.08.2016 again with only one Blank:

fig.1: The Average over replicates of fluorescence in RFU. Measured was the fluorescence of coated GFP (20 µg/ml) and the auto fluorescence of empty Wells as a negative control, by measuring from top with the Plate reader.


fig.2: The Average over replicates of fluorescence in RFU. Measured was the fluorescence of coated GFP (20 µg/ml) and the auto fluorescence of empty Wells as a negative control, by measuring from bottom with the Plate reader.

Prepared coating solutions with several concentrations:

• 20 µg/ml
• 28.7 µg/ml
• 41.8 µg/ml
• 35.6 µg/ml

Coat the wells with 50µl from prepared coating solution:

fig.3: The Average over replicates of fluorescence in RFU. Measured was the fluorescence of coated GFP (20 µg/ml, 28.7 µg/ml, 41.8 µg/ml and 35.6 µg/ml) and the auto fluorescence of empty Wells as a negative control, by measuring from top with the Plate reader.


fig.4: The Average over replicates of fluorescence in RFU. Measured was the fluorescence of coated GFP (20 µg/ml, 28.7 µg/ml, 41.8 µg/ml and 35.6 µg/ml), the auto fluorescence of empty Wells as a negative control and the fluorescence of GFP (20 µg/ml, 28.7 µg/ml, 41.8 µg/ml and 35.6 µg/ml) in solution as positive control, by measuring from top with the Plate reader.

Coated

1. Used Greiner MID bind plate
2. Spores: WT, B53, B54; Used 50µl/well, for Spores was purified by washing with PBS, to remove the medium.
3. used GFP coating concentrations of: 40µg/ml, 45µg/ml, 60µg/ml, 70µg/ml, 80µg/ml, 90µg/ml; used 50µl/well
4. m.Cherry: 20µg/ml, 40µg/ml, 80µg/ml

coted over night by 4°C in the fridge.

1. Washed plate for previous day twice with 200µl PBS
2. Incubated for 2h at room temperature
3. Blocked with 200µl 5% nonfat dry milk in PBS
4. Incubated for 2h at room temperature

Measured the Fluorescents at two different focal heights:
22mm:

fig.5: Average of fluorescence over replicates of samples (B53_coated 18mill., B53_solution 18mill., B53_coated 9mill., B53_solution 9mill., B53_coated 1.8mill., B53_solution 1.8mill., B54_cated 18mill., B54_solution 18mill., B54_coated 9mill., B54_solution 9mill., B54_coated 1.8mill., B54_solution 1.8mill., GFP_coated 20µg/ml, GFP_coated 35.6µg/ml, GFP_coated 40µg/ml, GFP_coated 45µg/ml, GFP_coated 60Mg/ml, GFP_coated 70µg/ml, GFP_coated 80µg/ml, GFP_coated 90µg/ml, m.Cherry_coated 20µg/ml, m.Cherry_coated 40µg/ml, m.Cherry_coated 80µg/ml, WT_coated 18mill.) and an empty well as negative control, at a focal height of 22mm.

2mm:

fig.6: Average of fluorescence over replicates of samples (B53_coated 18mill., B53_solution 18mill., B53_coated 9mill., B53_solution 9mill., B53_coated 1.8mill., B53_solution 1.8mill., B54_cated 18mill., B54_solution 18mill., B54_coated 9mill., B54_solution 9mill., B54_coated 1.8mill., B54_solution 1.8mill., GFP_coated 20µg/ml, GFP_coated 35.6µg/ml, GFP_coated 40µg/ml, GFP_coated 45µg/ml, GFP_coated 60Mg/ml, GFP_coated 70µg/ml, GFP_coated 80µg/ml, GFP_coated 90µg/ml, m.Cherry_coated 20µg/ml, m.Cherry_coated 40µg/ml, m.Cherry_coated 80µg/ml, WT_coated 18mill.) and an empty well as negative control, at a focal height of 2mm.

Scheme:

fig.7: Pipette scheme for the measuring of fluorescence at the surface by several concentrations of GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml) and two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54). The shaded wells shown dried wells with PBS, were the solution of GFP/Spores was dried by 37°C in the incubator.

1. took from B54 and B53 aliquot 108µl (108 mill. Spores) and 54µl (54 mill. Spores)
   
2. washed them by spinning down at 13000rct for 13min and resuspend the pellet in PBS, three times
3. resuspend final pellet in 300µl PBS -pipetted 50µl solution into the shaded wells (scheme)
4. incubated at 37°C for 5,5h
   

Measures the plate by different focal height and gain values:

• Focal height 22mm, gain 1740
• Focal height self-adjusted at 3.6mm, gain 1740
• Focal height 3.6mm, gain 2960

fig.8: Average of relative fluorescence over Replicates of dried in PBS, GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. It was used a setting with a focal height of 22mm and gain value of 1740.


fig.9: Average of relative fluorescence over Replicates of dried in PBS, GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. It was used a setting with a focal height of 3.6mm and gain value of 1740.


fig.10: Average of relative fluorescence over Replicates of dried in PBS, GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. It was used a setting with a focal height of 3.6mm and gain value of 2960.

fig.11: Average of relative fluorescence over Replicates with PBS on top GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. It was used a setting with a focal height of 22mm and gain value of 1740.


fig.12: Average of relative fluorescence over Replicates with PBS on top GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. It was used a setting with a focal height of 3.6mm and gain value of 1740.


fig.13: Average of relative fluorescence over Replicates with PBS on top GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. It was used a setting with a focal height of 3.6mm and gain value of 2960.

scheme:

fig.14: Pipette scheme for the measuring of fluorescence at the surface by several concentrations of GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml) and two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54). The shaded wells shown dried wells with H2O, were the solution of GFP/Spores was dried by 37°C in the incubator.

• Prepared dilution series of GFP and Spores in H2O as in the scheme
• Pipetted 100µl per well and incubated it at 37°C for 12h 40min.

Measured the plate with three different settings:
* Focal height 22mm, gain 1740 * Focal height 3.6mm, gain 1740 * Focal height 3.6mm, gain 2960

Focal height 22mm, gain 1740:

fig.15: Average of relative fluorescence over Replicates of dried in H2O, GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. It was used a setting with a focal height of 22mm and gain value of 1740.

Focal height 3.6mm, gain 1740:

fig.16: Average of relative fluorescence over Replicates of dried in H2O, GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. It was used a setting with a focal height of 3.6mm and gain value of 1740.

Focal height 3.6mm, gain 2960:

fig.17: Average of relative fluorescence over Replicates of dried in H2O, GFP (20, 15, 10, 5, 1, 0,1, 0,01, 0,001 µg/ml), two different amounts (18 mill., 9 mill.) of spores of two different types (B53, B54) and empty wells as negative control. It was used a setting with a focal height of 3.6mm and gain value of 2960.

• Coated free wells on the plate where GFP dried in with PBS, used wells D5 – D12, H5 – H12 and D1 as blank
• Coated concentrations of: 20µg/ml (D5 – D7), 15µg/ml (D8 – D10), 10µg/ml (D11, D12 and H5), 5µg/ml (H6 – H8)
• Coated with 50µl
• Incubated for 2h at room temperature
• Washed wells twice with 200µl PBS
• Blocked with 200µl 5% nonfat dry milk in PBS
• Incubated for 2h at room temperature
• Washed the wells twice with 200µl PBS

Measured the emission of the fluorescence with several settings:
Focal height 22mm, gain 1740
Focal height 3.6mm, gain 174
Focal height 3.6mm, gain 2960

Focal height 22mm, gain 1740:

fig.18: Measured the relative fluorescence over replicates of coated GFP (20µg/ml, 15µg/ml, 10µg/ml, 5µg/ml) and empty wells as negative control, at focal height 22mm with a gain value of 1740.

Focal height 3.6mm, gain 1740:

fig.19: Measured the relative fluorescence over replicates of coated GFP (20µg/ml, 15µg/ml, 10µg/ml, 5µg/ml) and empty wells as negative control, at focal height 3.6mm with a gain value of 1740.


fig.20: Measured the relative fluorescence over replicates of coated GFP (20µg/ml, 15µg/ml, 10µg/ml, 5µg/ml) and empty wells as negative control, at focal height 3.6mm with a gain value of 2960.

1. Used Greiner HIGH bind plate
2. Coating concentration of GFP20µg/ml
3. Coating volume 50µl
4. Incubated for 4h at room temperature

1. Washed coated wells twice with 200µ PBS
2. Blocked with 200µl 5% nonfat dry milk in PBS
3. Incubated for 2h at room temperature
4. Washed wells twice with 200µl PBS

Measured with a focal height of 3.6mm and a gain value of 1740

fig.20: Average of relative fluorescence units over replicates of coated GFP (20µg/ml) and empty wells as negative control.

scheme:

fig.21: washed coated GFP with different detergents (SDS, Tx100, Tween20, Shampoo1 (Syoss OLEO 21) and Shampoo 2 (Herbal Essences Verwöhnende Feuchtigkeit), one group was washed with 2% SDS as positive control, empty was washed with PBS and wasn’t coated with GFP, the positive control was washed with PBS.

Concentration for each detergent:
SDS: 2%; 0,1%; 0,01%; 0,001%; 0,0001%
Triton: 0,1%; 0,01%; 0,001%; 0,0001%
Tween: 0,1%; 0,01%; 0,001%; 0,0001%
Shampoo 1: 0,1%; 0,01%; 0,001%; 0,0001%
Shampoo 2: 0,1%; 0,01%; 0,001%; 0,0001%

1. Washed the plate after the scheme with 200µl detergent and shook for 2min, pipetted out.
2. Measured the fluorescence of GFP
3. Washed with PBS
4. Measured the fluorescence of GFP
5. Washed again with 200µl detergent and shook for 2min, pipetted out
6. Measured the fluorescence of GFP
7. Washed with PBS
8. Measured the fluorescence of GFP

Setting:gfg_highbind_deterg_settings.xlsx

Setting:gfp_highbind_deterg_pbs_settings.xlsx

Setting:gfp_highbind_againdet-settings.xlsx

Setting:gfp_highbind_againdet_pbs-settings.xlsx

Prepared different concentrations of detergent:

• SDS: 2%; 1,5%; 1%; 0,5%; 0,1%
• Triton: 1,5%; 1%; 0,5%; 0,1%
• Tween: 1,5%; 1%; 0,5%; 0,1%
• Shampoo 1: 1,5%; 1%; 0,5%; 0,1%
• Shampoo 2: 1,5%; 1%; 0,5%; 0,1%

For washing used plate and scheme from 19.06.2016 with different concentrations.
washed the coated wells with the detergents, pipetted 200µl detergent and shook for 2min, pipetted out.
Measured the fluorescence emission after washing with detergent.
Measured the fluorescence emission after washing with PBS.

after washing with detergents
washed with PBS afterwards

fig.24: RFU difference between last measurement of the plate (washed again with PBS) and washed with detergents: SDS (2%; 1,5%; 1%; 0,5%; 0,1%), Triton: 1,5%; 1%; 0,5%; 0,1%), Tween (1,5%; 1%; 0,5%; 0,1%), Shampoo 1 (1,5%; 1%; 0,5%; 0,1%), Shampoo 2 (1,5%; 1%; 0,5%; 0,1%), SDS 2% was used as positive control. Used a focal height of 3.6mm and a gain value of 1740.

Prepared dilution of GST-Stock (5Units/ml):

• 1U/ml
• 0.75U/ml
• 0.5U/ml
• 0.1U/ml
• 0.02U/ml
• 0.001U/ml
  

Volume assay cocktail 1.5ml (15µlCDNB [100mM] + 15µl GSH [100mM] + 1470µl PBS).

Plate scheme:

fig.25: scheme for measuring the activity of different GST concentrations (1U/ml, 0.75U/ml, 0.5U/ml, 0.1U/ml, 0.02U/ml, 0.001U/ml), Empty (PBS and Assay cocktail), Blank (PBS and Assay cocktail).

start the reaction with 30µl GST as in the scheme (Abb.27), in blank and empty (control) was added 30µl PBS.
Setrtings:protocol_gst_run_1_.xlsx

All data and graphs were collected in an Excel file: freiburg_2016_gst_run_1.xlsx

prepared samples:

• 0.3U (2x 0.15U) 0.15U (90µl of 5U/ml stock)
• 0.25U (2x 0.125U) 0.125U (25µl of 5U/ml stock + 5µl PBS)
• 0.2U (2x 0.1U) 0.1U (20µl of 5U/ml stock + 10µl PBS)
• 0.15U (2x 0.075U) 0.075U (15µl of 5U/ml stock + 15µl PBS)
• 0.1U (2x 0.05U) 0.05U (10µl of 5U/ml stock + 20µl PBS)
• 0.05U (2x 0.025U) 0.025U (5µl of 5U/ml stock + 25µl PBS)
• 0.02U (2x 0.01U) 0.01U (2µl of 5U/ml stock + 28µl PBS)

volume auf each sample contains 30µl.
Prepared assay cocktail (1470µl PBS + 15µl GSH [100mM] + 15µl CDNB [100mM]).
Pipetted GST – samples after the scheme (fig.26)

Scheme:

fig.26: pipetting scheme of GST-amount (0.15U, 0.125U, 0.1U, 0.075U, 0.05U, 0.025U, 0.01U), white empty (Assay cocktail and PBS) in black Blank (Assay cocktail and PBS).

• Started the reaction with 90µl assay cocktail in each well
• Put it immediately in the plate reader
• Used measurement settings:protocol_gst_run_2_.xlsx
  

All data and Graphs were collected in an excel file:freiburg_2016_18.09.16_gst-assay_02_.xlsx

Repeated the assay of GST – Assay 02
Pipetting scheme as in GST – Assay 02 (fig.26)

• Start the reaction with 90µl assay cocktail in each well
• Used settings as shown in the reader protocol: protocol_gst_run_3.xlsx

All graphs were collected in an excel file: freiburg_2016_gst_run3.xlsx

Repeat the assay depending on the results of GST – Assay 03 (Freiburg 2016_gst_run3.xlsx). increased the measuring time up to 10min and pipetted on ice.
Pipetted after scheme shown in fig.26.

• Start the reaction with 90µl assay cocktail in each well
• Put it immediately in the plate reader
• Used settings as shown in the reader protocol: protocol_gst_run_4.xlsx

All graphs were collected in an excel file: freiburg_2016_gst_run4.xlsx

Scheme:

fig.27: scheme of detergent screening for SDS, Tx100, Tween20, Shampoo1 and Shampoo2, as positive control was used a solution of 2% SDS, for negative control was used a wash solution of PBS without detergents.

1. Prepared GFP coating solution (20µg/ml)
2. Coated the plate after the scheme (fig.27) with coating volume of 100µl
3. Incubated for 2h at room temperature
4. Washed the wells twice with 200µl PBS
5. Blocked wells with 200µl 5% nonfat dry milk in PBS
6. Incubated for 2h at room temperature
7. Washed wells twice with 200µl PBS

Preparation of detergents for screening: Prepared stock solutions of 20% and 40%
- Prepared samples of 1ml for each the three cycles (except Block three there were only two cycles) in each block:

• Block 1: 2, 4, 6, 8% solutions of Tx100, Tween20, Shampoo1 (SH1) and Sampoo2 (SH2); for SDS used concentrations 1.5, 0.25, 1, 0.5 and 0.1% solutions
• Block 2: 12, 14, 16,1 8% solutions of Tx100, Tween20, Shampoo1 (SH1) and Sampoo2 (SH2); for SDS used concentrations 1.5, 0.75, 1, 0.5 and 0.1% solutions
• Block 3: 32, 34,3 6, 38% solutions of Tx100, Tween20, Shampoo1 (SH1) and Sampoo2 (SH2); for SDS used concentrations 1.5, 1.25, 1, 0.5 and 0.1% solutions

Block 1:

1. Washed with concentrations of 2, 4, 6, 8% solutions of Tx100, Tween20, Shampoo1 (SH1) and Sampoo2 (SH2); for SDS used concentrations 1.5, 0.25, 1, 0.5 and 0.1%, for negative control used PBS and for positive control 2% SDS. Wash solution volume took 200µl.
2. washed with PBS twice afterwards
3. pipetted out the whole volume which was left in the wells
4. measured the fluorescence
5. repeated it for a total of 3 cycle

Block 2:

1. Washed with concentrations of 12,14, 16, 18% solutions of Tx100, Tween20, Shampoo1 (SH1) and Sampoo2 (SH2); for SDS used concentrations 1.5, 0.75, 1, 0.5 and 0.1%, for negative control used PBS and for positive control 2% SDS. Wash solution volume took 200µl.
2. washed with PBS twice afterwards
3. pipetted out the whole volume which was left in the wells
4. measured the fluorescence
5. repeated it for a total of 3 cycle

Block 3:

1. Washed with concentrations of 32,34, 36, 38% solutions of Tx100, Tween20, Shampoo1 (SH1) and Sampoo2 (SH2); for SDS used concentrations 1.5, 1.25, 1, 0.5 and 0.1%, for negative control used PBS and for positive control 2% SDS. Wash solution volume took 200µl.
2. washed with PBS twice afterwards
3. pipetted out the whole volume which was left in the wells
4. measured the fluorescence
5. repeated it

Data and figures were collected in an excel file:freiburg_2016_30.08.16_detergengt_screening.xlsx

Fig.28: wash efficiency of detergents in an overview.

Plate name W5
coated the plate with GFP after coating protocol, used scheme from fig.29

scheme:

Fig.29: washing scheme with several concentrations of detergents: SDS: 1.25, 1, 0.75, 0.25%; Tween20: 22, 24, 26, 28, 32, 24, 26, 28%; SH1: 12, 14, 16 ,18, 22, 24, 26, 28%. For neg. contr. used 2% SDS, pos. contr. was washed with PBS without detergent.

stock solution 40%

12% | 300µl stock solution + 700µl PBS
14% | 350µl stock solution + 650µl PBS
16% | 400µl stock solution + 600µl PBS
18% | 450µl stock solution + 550µl PBS
22% | 550µl stock solution + 450µl PBS
24% | 600µl stock solution + 400µl PBS
26% | 650µl stock solution + 350µl PBS
28% | 700µl stock solution + 300µl PBS
32% | 800µl stock solution + 200µl PBS
34% | 850µl stock solution + 150µl PBS
36% | 900µl stock solution + 100µl PBS
38% | 950µl stock solution + 50µl PBS

used setting for measuring the fluorescence form coated GFP and washed plate as shown in the protocol:

Detailed figures collected in an excel file:

Fig.30: overview of the wash efficiency of detergents: SDS: 1.25, 1, 0.75, 0.25%; Tween20: 22, 24, 26, 28, 32, 24, 26, 28%; SH1: 12, 14, 16 ,18, 22, 24, 26, 28%. For neg. contr. used 2% SDS, pos. contr. was washed with PBS without detergent.

repeated the assay depending on the results of GST – Assay 04 (excel file: Freiburg 2016gst_run4.xlsx.

scheme:

fig.26: pipetting scheme of GST-amount (0.15U, 0.125U, 0.1U, 0.075U, 0.05U, 0.025U, 0.01U), white empty (Assay cocktail and PBS) in black Blank (Assay cocktail and PBS).

1. prepared the samples on ice
2. incubated the plate before started in the fridge for 5min.
3. prepared samples after the scheme shown in fig.26

1. started the reaction with 90µl assay cocktail in each well
2. put it immediately in the plate reader
3. used settings as shown in the reader protocol: protocol_gst_run_5.xlsx

All data and graphs were collected in an excel file: freiburg_2016_gst_run5.xlsx

Preparation as like in GST – Assay 05. Took a little more time to start the measuring after mixing enzyme and assay cocktail
Used pipetting scheme as shown in fig.26.

Used setting as shown in the reader protocol: protocol_gst_run_6.xlsx

All data and graphs were collected in an excel file: freiburg_2016_gst_run6.xlsx

Repeated like as in assay 05, but all was pipetted on ice. Used pipetting scheme as shown in fig.26.

Start the reaction with 90µl assay cocktail from low amount of GST concentration to high, for reducing the time delay the assay cocktail was piped in directly next to the plate reader

All data and Graphs were collected in an excel file: freibur_2016_gst_run7.xlsx

Preparations like as in the other Assays before (GST – Assay 07) after pipetted and incubate for 5min in the fridge put the plate on a shaker.

started the reaction with 90 µl Assay cocktail while shaking at 300rpm and temperature (temperature of the block) of 12°C.
Put it then directly into the reader.
protocol: protocol_gst_run_8.xlsx

graphs were collected in an excel file: freiburg_2016_gst_run8.xlsx

* as in GST-Assay 8 * increased measuring time up to 20min in the measuring settings

Started the reaction like in GST-Assay 8, used settings as shown in the protocol: protocol_gst_run_9.xlsx

All data and graphs were in an excel file: freiburg_2016_gst_run9.xlsx

• 0.15U (90µl of 5U/ml stock)
• 0.075U (15µl of 5U/ml stock + 75µl PBS)
• 0.025U (5µl of 5U/ml stock + 85µl PBS)
• 0,005U (3µl of 5U/ml stock + 87µl PBS)

Assay cocktail: 15µl CDNB + 15µl GSH + 1470µl PBS.
Used 30µl enzyme solution and 90µl Assay cocktail per well, for Blank and Empty used 30µl PBS and Assay cocktail.

Scheme:

fig.31: Pipetting scheme for GST – Assay wit 0.15, 0.075, 0.025 and 0.005 Units of GST and Assay cocktail, for wells empty and blank used instead of GST solutions PBS and Assay cocktail.

Assay cocktail wasn’t enough for all wells, concentration of 0.15U without assay cocktail.

Used setting as shown in reader protocol:

Data and Graphs were collected in an excel file:

Repeated GST – Assay 10 with more assay cocktail, scheme fig.31
1,7ml: 17µl CDNB + 17µl GSH + 1666µl PBS

Used setting as shown in reader protocol:

Data and Graphs were collected in an excel file:

Repeated measurement of GST – Assay 11 with different measurement settings.
Used scheme the same scheme fig.31

Used setting as shown in reader protocol:

Data and Graphs were collected in an excel file:

Coated plate after the scheme
Prepared nanobody solution: Concentration nanobody stock solution 0.48mg/ml, set up dilution of 1.8ng/µl
Scheme:

fig.32: washing scheme for washing with nanobody and detergents. As control were used: GFP washed with PBS (negative control 1), washed with nanobody in PBS (negative control 2), 2%SDS (positive control). As reference were empty wells not coated and unwashed, same as the blank. Used detergent samples: SDS: 1, 0.75, 0.5, 0.25% solutions with nanobody, Tween20: 22, 24, 26,28% solutions with nanobody.

1. Measured the fluorescence of coated GFP
2. Washed with 1µl nanobody and 200µl concentrations of detergents: SDS: 1.5, 1, 0.5, 0.25%, Tween20: 22, 24, 26, 28%, SH1: 12, 14, 16, 18%
3. Incubated it for 5min
4. Shook it for 5min at 300rpm
5. washed with PBS twice afterwards
6. pipetted out the whole volume which was left in the wells
7. measured the fluorescence, used same settings as measured the fluorescence of coated GFP:

Detailed column charts were collected in an excel file: washing_with_nanobody_eff..xlsx


fig.33: overview of the efficiency of washing with nanobody in combination with detergents. SDS: 1, 0.75, 0.5, 0.25% solutions with nanobody, Tween20: 22, 24, 26,28% solutions with nanobody. Controls GFP washed with PBS (negative control 1), washed with nanobody in PBS (negative control 2), 2%SDS (positive control).

Scheme:

fig.34: pipetting scheme of GST – Assay, used GST amount of: 0.0001, 0.00001, 0.000001, 0.0000001 Units, for empty and blank was used PBS instead of GST.

Measurement settings as in the reader protocol:14.09.16_gst-assay_settings.xlsx

All data and graphs were collected in an excel file: freiburg_2016_14.09.16_gst-assay_-1.xlsx

Platename: WN2
Scheme:
Fig.35: washing scheme for washing with nanobody and detergents. As control were used: coated GFP not washed (negative control 1), GFP washed with PBS (negative control 2), washed with nanobody in PBS (negative control 3), 2%SDS (positive control). As reference were empty wells not coated and unwashed, same as the blank. Used detergent samples: SDS: 1, 0.75, 0.5, 0.25% solutions with nanobody, Tween20: 22, 24, 26,28% solutions with nanobody.

1. coated plate with GFP (coating concentration 20µg/ml)
2. dilute 1µl nanobody stock solution (0.48mg/ml) in prepared samples 1ml detergents: SDS: 1, 0.75, 0.5, 0.25, Tween20: 22, 24, 26, 28%, SH1: 12, 14, 16, 18%

1. Measured the fluorescence of coated GFP
2. Washed with 200µl detergents: SDS: 1.5, 1, 0.5, 0.25% (Row A and B with nanobody, row C and D without nanobody), Tween20: 22, 24, 26, 28% (Row A and B with nanobody, row C and D without nanobody), SH1: 12, 14, 16, 18% (Row E and F with nanobody, row G and H without nanobody)
3. Incubated it for 5min
4. Shook it for 5min at 300rpm
5. washed with PBS twice afterwards
6. pipetted out the whole volume which was left in the wells
7. measured the fluorescence
8. used same settings as measured the fluorescence of coated GFP:
   

Detailed column charts were collected in an excel file:freiburg_2016_16.09.16_washing_nanobody_02_gfp_master.xlsx


Fig.36: Overview of the wash efficiency between with and without nanobody for: SDS, SDS + nanobody, Tween20, Twenn20 + nanobody, SH1, SH1 + nanobody.

Repeat the GST – Assay 13 with the same scheme (fig34).
Concentration of samples:

• 0,0001U (3µl 0,1U/ml stock + 87µl PBS)
• 0,00001U (3µl 0,01U/ml stock + 87 PBS)
• 0,000001U (3µl 0,001U/ml stock + 87 PBS)
• 0,0000001U (3µl 0,0001U/ml stock + 87 PBS)

Assay cocktail: 15µl CDNB + 15µl GSH + 1470µl PBS.
Pipetted the GST after the scheme (30µl per well), used for empty wells and blank instead of GST solution PBS.

Start the reaction with 90µ assay cocktail from low concentration to high concentration, pipetted directly next to the plate reader to recuse the time delay.
Measured the absorbents with settings as described in the measurement protocol: 17.09.16_gst-assay_01_settings.xlsx

Repeated the GST – Assay 14
settings: 17.09.16_gst-assay_02_settings.xlsx

Changed setting for the measurement: shook in plate reader before each cycle at 500rpm 1s.

Scheme:

fig.37: GST – Assay pipetting scheme for GST concentrations of 0.005 and 0.0000001 Units. Empty as negative control with PBS instead of GST, blank as the same conditions like empty.

prepared

• samples of 0.005 and 0.0000001U
• assay – cocktail (14µl CDNB + 14µl GSH + 1372µl PBS)
• pipetted GST-samples after the scheme and PBS for the controls (30µl)

start the reaction with 170µl Assay cocktail. Used new setting as in the reader protocol described: 17.09.16_gst-assay_03_settings.xlsx

All data and graphs of GST - Assay 14 - 16 were collected in an excel file: freiburg_2016_17.09.16_gst-assay_master.xlsx

Doubled the amount of substrate
Scheme:

Fig.38: Pipetting scheme for GST – Assay with 0.1, 0.025, 0.005 and 0.001 Units (U), empty wells and blank measured with PBS instead of GST as control.

GST – samples:

• 0.1U (60µl of 5U/ml stock + 30µl PBS)
• 0.025U (5µl of 5U/ml stock + 87µl PBS)
• 0.005U (3µl of 5U/ml stock + 85µl PBS)
• 0,001U (3µl of 1U/ml stock + 87µl PBS)

Assay cocktail: 30µl CDNB + 30µl GSH + 1440µl PBS.
Pipet GST – samples and PBS after the scheme (fig.38).

Start the reaction with 90µl assay cocktail
Start measuring with settings which are described in the protocol: 18.09.16_gst-assay_01_settings.xlsx

Data and graphs were collected in an excel file:18.09.16_gst-assay_01.xlsx

Scheme:

fig.39: Pipetting scheme for GST – Assay with 0.1, 0.05 Units (U), empty wells and blank measured with PBS instead of GST as control, grey wells are not used.

Increased the measuring time up to 33 minutes.

GST – Samples:

• 0,1U
• 0,05U (30µl of 5U/ml stock + 60µl PBS)
• 0,025U
  

Assay cocktail

Start reactions with 90µl assay cocktail Used setting as decried in the measurement protocol of the reader: 18.09.16_gst-assay_02_settings.xlsx

Data and graphs were collected in an excel file: freiburg_2016_18.09.16_gst-assay_02_-1.xlsx

Scheme:

fig.40: Pipetting scheme for GST – Assay with 0.1, 0.05 Units (U), empty wells and blank measured with PBS instead of GST as control, grey wells are not used.

GST concentrations:

• 0,1U (80µl 5U/ml stock + 40µl PBS)
• 0,05U (40µl 5U/ml stock + 80µl PBS)

Assay cocktail: 18µl CDNB + 18µl GSH + 1164µl PBS
Changed shook time before the first cycle in the setting to 200rpm for 1s
Piped the samples after the scheme in fig.42

Start the reactions with 90µl PBS and start measurement with the setting as described in the plate reader protocol: 19.09.16_gst-assay_01_settings.xlsx

Repeat the GST – Assay 19 in addition with one well of enzyme without assay cocktail
Scheme:

Fig.41: Pipetting scheme for GST – Assay with 0.1, 0.05 Units (U), empty wells and blank measured with PBS instead of GST as control, En well which contains only enzyme without assay cocktail, grey wells are not used.

GST concentrations:

• 0,1U (80µl 5U/ml stock + 40µl PBS)
• 0,05U (40µl 5U/ml stock + 80µl PBS)

Assay cocktail: 18µl CDNB + 18µl GSH + 1164µl PBS
For En (fig.41) used 30µl of 1U GST solution
Piped the samples after the scheme in fig.41

Start the reactions with 90µl PBS and start measuremen.

All data and graphs of GST - Assay 19 and 20 were collected in an excel file: freiburg_2016_19.09.16_gst-assay_master-1.xlsx

Scheme:

Fig.42: Pipetting scheme for GST – Assay with 0.1, empty wells and blank measured with PBS instead of GST as control, grey wells are not used.

Sample of 0.1U and assay cocktail: 70µl CDNB + 70µl GSH + 560µl PBS, increased amount of substrates.

Start the reaction with 90µ assay cocktail.
Start the measurement wit settings as before, which are described also in the reader protocol: 23.09.16_gst-assay_01_settings.xlsx

Reduced amount of substrates from 70µl to 50µl and increased volume from 90µ to 110µl used blank of GST – Assay 21 (scheme fig.42)
Scheme:

Fig.43: Pipetting scheme for GST – Assay 22. 0.1Units GST for sample wells and as referents Empty which contains PBS and assay cocktail.

• Prepared 0.1U samples of GST and assay cocktail: 50µl CDNB + 50µl GSH + 900µl PBS
• pipetted 30µl GST and PBS after the scheme (fig.43)

Start the reactions with 110µl assay cocktail
started measurement with setting which are described in the plate reader protocol:23.09.16_gst-assay_02_settings.xlsx

Result: All data and graphs of GST - Assay 22 and 23 were collected in an excel file: freiburg_23.09.16_gst-assay_master-1.xlsx