Difference between revisions of "Team:Freiburg/NotebookTargeting"

Bacillus subtilis strains used:

Slide preparation: GOPTS

Surface activation of glass slides by plasma generator, coated with GOPTS 3-glycidyloxypropyl trimethoxysilane, washed with acetone and then stored for up to two weeeks.

1. labeled with a diamond pen
2. washed with EtOH then ddH2O
3. dried slides with compressed air
4. activation of surface with plasma generator
5. incubation with GOPTS (overnight)

Buffer for anti-GFP nanobody (10 ml)

• 61mg Tris
• 87mg NaCl
• 1ml Glycerin
• 2,5 ml Imidazole(1M)
• fill up with ddH2O to 10 ml

Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56

1. incubation with nanobody (0.5µl, 330µg/ml, overnight, 4°C)

Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56

1. blocked with BSA(2%) (150µl/spot, 30 min)
2. washed with PBS(1x)
3. washed with imidazole (20mM) in PBS(1x)
4. washed with PBS(1x)
5. washed with ddH2O
6. dried with compressed air
7. incubation with B52-B56 (100µl/spot, 120 min)
8. incubation with Mowiol DABCO(1,4-Diazabicyclo[2.2.2]octane) (5µl/spot, overnight), with second glass slide on top

Sample preparation 1: GOPTS + anti-GFP nanobody + B52-B56

1. edges sealed with nail polish

Fluorescence microscopy 1 (Olympus 1×71): GOPTS + anti-GFP nanobody + B52-B56

–> pictures inconclusive: wrong coverslips (second glass slide), no sufficient controls

Sample preparation 2: GOPTS + anti-GFP nanobody + B52-56

1. incubation with anti-GFP nanobody (5µl/spot, 330 µg/ml, 120 min)
2. blocked with BSA(2%) (90 min)
3. rinsed with PBS(1x)
4. stored in PBS(1x) (for 9 days)

Sample preparation

1. added nanobody to GOPTS plate (300 µg/ml, 5µl)
2. tried to pipette rest nanobody down, but didn´t work
3. blocked complete slide with BSA(2%)

Sample preparation 2: GOPTS + anti-GFP nanobody + B52-56

1. washed slides with ddH2O
2. incubation with B52, B53, B54, B55, B56 (50µl/spot, overnight)

–> FACS-analysis has shown that only B53 and B54 show fluorescence linked to GFP

Preparation of samples 3: B53-B55

1. B53, B54, B55 (10µl/spot) on uncoated glass slide
2. Mowiol DABCO (5µl/spot)
3. covered with cover slip

Fluorescence microscopy 2 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B52-56

–> fluorescence signal of B53 and B54

Fluorescence microscopy 3 (Olympus 1×71): B53-B55

–> fluorescence signal of positive controls (B53 and B54) but no signal of spores bound to GOPTS

Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54

1. incubation with anti-GFP nanobody (5µl, 330µg/ml, 2d)

Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54

1. blocked with BSA(4%) (overnight)

Sample preparation 4: GOPTS + anti-GFP nanobody + B53/B54

1. rinsed and washed with PBS(1x) (10 min, 10rpm)
2. rinsed with water ddH2O
3. dried with compressed air
4. incubation with Mowiol DABCO (5µl/spot, overnight)

Fluorescence microscopy 4 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53/B54

–> spores appear to not have been bound to nanobody

Sample preparation 5: GOPTS + anti-GFP nanobody + B53-56/GFP

1. incubation with anti-GFP nanobody (5µl, 330µg/ml, 60 min)
2. rinsed and blocked with BSA(4%) (30 min)
3. incubation with B53, B54, B55, B56, GFP (5µl/spot, 60 min)
4. rinsed and washed with PBS(1x) (10 min, 10 rpm)
5. rinsed with water ddH2O
6. dried with compressed air
7. incubation with Mowiol DABCO (5µl/spot, overnight)

Fluorescence microscopy 5 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53-56/GFP

–> spores have not been bound

Sample preparation 6: anti-GFP nanobody + spores (WT, B53, B54, B56) (in solution)

1. incubation of anti-GFP nanobody (Alexa647-conjugated) (1µl, 330µg/ml, 60 min) + spores (WT, B53, B54, B56) (400µl, 60 min, 4°C)
2. centrifuged (13400 rpm, 2 min)
3. washed with PBS(1x) (1 ml)
4. repeated steps 2 and 3
5. centrifuged (13400 rpm, 2 min)
6. resuspended in PBS(1x) (400µl)

Fluorescence microscopy 6 (Nikon C2+ confocal microscope): anti-GFP nanobody + spores (WT, B53, B54, B56) in solution

–> no introduction to confocal mode: nanobody not observable

Sample preparation 7: GOPTS + anti-GFP nanobody + B53/B54

1. incubated with nanobody (1µl, 330µg/ml, 60 min)
2. rinsed and blocked with BSA(4%) (60 min)
3. rinsed and washed twice with PBS(1x) (10 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubated with spores (10µl/spot, 120 min)
7. rinsed and washed twice with PBS(1x) (10 min, 10rpm)
8. rinsed with ddH20
9. dried with compressed air
10. incubation with Mowiol DABCO (5µl, overnight)

Washing test A: GOPTS + anti-GFP nanobody + B53

1. incubation with nanobody (1µl/mg, 300µg/ml, overnight, 4°C)

Washing test A: GOPTS + anti-GFP nanobody + B53

1. rinsed and blocked with BSA(2%) (60 min, 10 rpm)
2. rinsed with PBS(1x), washed PBS twice (10 min, 10 rpm)
3. rinsed with ddH2O
4. dried with compressed air
5. incubation with B53 (10µl/spot, overnight)

Washing test A: GOPTS + anti-GFP nanobody + B53

1. rinsed and washed (see table below)
2. rinsed with ddh2O
3. dried with compressed air
4. incubated with Mowiol DABCO (5µl, overnight)

Fluorescence microscopy 7 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53/B54

–> spores have not bound to nanobody

Fluorescence microscopy A (Nikon C2+ confocal microscope): Washing test: GOPTS + anti-GFP nanobody + B53

–> spores have not bound to BSA

Sample preparation 8: GOPTS + anti-GFP nanobody + GFP

1. incubation with nanobody (see table below, 60 min)
2. rinsed and blocked with BSA(4%) (60 min)
3. rinsed and washed with PBS(1x) (5 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubated with GFP (see table below, 1.35mg/ml, 20 min)
7. rinsed and washed with PBS(1x) (10 min, 10rpm)
8. rinsed with ddH20
9. dried with compressed air
10. incubation with Mowiol DABCO (5µl, overnight)
    

Fluorescence microscopy 8 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP

–> GFP not bound, GOPTS plates too old

Sample preparation 9: GOPTS + GFP

1. incubation with GFP (1.35mg/ml, volume/incubation: see table below)
2. rinsed and blocked with BSA(4%) (30 min)
3. rinsed and washed with PBS(1x) (10 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubation with Mowiol DABCO (5µl, overnight)

Sample preparation 10: GOPTS + anti-GFP nanobody + B53

1. incubation with nanobody (1µl, 330µg/ml, 60 min)
2. rinsed and blocked with BSA(4%) (60 min)
3. rinsed and washed with PBS(1x) (10 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubated with spores (10µl/spot, 30 min)
7. rinsed and washed with PBS(1x) (10min, 10rpm)
8. rinsed with ddH20
9. dried with compressed air
10. incubation with Mowiol DABCO (5µl, overnight)

Washing test B: GOPTS + anti-GFP nanobody + B53

1. incubation with nanobody (1µl, 330µg/ml, 60 min)
2. rinsed and blocked with BSA(4%) (60 min)
3. rinsed and washed with PBS(1x) (10 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubated with spores (10µl/spot, 30 min)
7. rinsed and washed with PBS(1x) (10 min, 10rpm)
8. rinsed with ddH20
9. dried with compressed air
10. incubation with Mowiol DABCO (5µl, overnight)

Fluorescence microscopy 9 (Nikon C2+ confocal microscope): GOPTS + GFP

–> strong fluorescence signal at some of the edges: incubation too long/volume too little, GFP has dried and could not have been washed away properly

• 1) GOPTS + GFP (1 µl, 10 min)
• 2) GOPTS + GFP (2 µl, 10 min)
• 3) GOPTS + GFP (5 µl, 10 min)
• 4) GOPTS + GFP (1 µl, 15 min)
• 5) GOPTS + GFP (2 µl, 15 min)
• 6) GOPTS + GFP (5 µl, 15 min)
• 7) GOPTS + GFP (1 µl, 20 min)
• 8) GOPTS + GFP (2 µl, 20 min)
• 9) GOPTS + GFP (5 µl, 20 min)

Fluorescence microscopy 10 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + B53

–> no spores appear to have bound to nanobody

Fluorescence microscopy B (Nikon C2+ confocal microscope): Washing test: GOPTS + anti-GFP nanobody + B53

–> no spores after washing, no binding between nanobody and B53?

Blocking test C: GOPTS + BSA/milk powder + GFP

1. blocking with BSA(4%)/milk powder(5%) (120 min)
2. rinsed and washed with PBS(1x) (10 min, 10 rpm)
3. incubation with GFP (1µl/spot, 1.35mg/ml, 20 min)
4. rinsed and washed with PBS(1x) (10 min, 10 rpm)
5. rinsed with ddH20
6. dried with compressed air
7. incubation with Mowiol DABCO (5µl, overnight)

Contamination Test D: BSA, PBS Mowiol DABCO

–> no contamination/pollution detected

Sample preparation 11: GOPTS + mCherry

1. incubated with mCherry (1µl, 2mg/ml, incubation: see table below)
2. rinsed and blocked with BSA(4%) (30 min)
3. rinsed and washed with PBS(1x) (10 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubated with Mowiol DABCO (5µl, overnight)

Sample preparation 12: GOPTS + anti-GFP nanobody + GFP

1. incubated with nanobody (5µl, 330µg/ml, 120 min)
2. rinsed and blocked with milk powder(5%) (60 min)
3. rinsed and washed with PBS(1x) (10 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubated with GFP (2µl, 1.35mg/ml, 20 min)
7. rinsed and washed with PBS(1x) (10 min, 10rpm)
8. rinsed with ddH20
9. dried with compressed air
10. incubated with Mowiol DABCO (5µl, overnight)

Fluorescence microscopy C (Nikon C2+ confocal microscope): Blocking test: GOPTS + BSA/milkpowder + GFP

–> No GFP signal after blocking with BSA and milk powder

Fluorescence microscopy 11 (Nikon C2+ confocal microscope): GOPTS + mCherry

–> no mCherry-signal, too weak (quantum yield of mCherry about 1/3 of that of GFP)

Fluorescence microscopy 12 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP

–> GFP has not bound to nanobody

Sample preparation 13: GOPTS + nanobody + GFP

1. incubated with nanobody (5µl, 330µg/ml, 60 min)
2. rinsed and blocked with milk powder(5%) (30 min)
3. rinsed and washed with PBS(1x) (10 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubated with GFP (2µl, 1.35mg/ml, 20 min)
7. rinsed and washed with PBS(1x) (10 min, 10rpm)
8. rinsed with ddH20
9. dried with compressed air
10. incubated with Mowiol DABCO (5µl, overnight)

Fluorescence microscopy 13 (Nikon C2+ confocal microscope): GOPTS + nanobody + GFP

–> no GFP signal detectable

Sample preparation 14: GOPTS + GFP

1. incubated with GFP (10µl, 1.35mg/ml, 30 min)
2. rinsed and blocked with milk powder(5%) (30 min)
3. rinsed and washed with PBS(1x) (10 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubated with Mowiol DABCO (5µl, overnight)

Mowiol was produced due to this protocol: https://www.carlroth.com/downloads/ba/en/0/BA_0713_EN.pdf

Fluorescence microscopy 14 (Nikon C2+ confocal microscope): GOPTS + GFP

–> spots with evenly distributed GFP

• 1) GOPTS + GFP
• 2) GOPTS + nanobody + GFP

Sample preparation 16: GOPTS + B53

1. incubated with B53 (5µl, 60 min)
2. rinsed and blocked with milk powder(4%) (30 min)
3. rinsed and washed with PBS(1x) (10 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubated with Mowiol DABCO (5µl, overnight)

Sample preparation 17: GOPTS + anti-GFP nanobody(Alexa647) + GFP

1. incubated with nanobody (5µl, 330µg/ml, 60 min)
2. rinsed and blocked with milk powder(5%) (30 min)
3. rinsed and washed with PBS(1x) (10 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubated with GFP (2µl, 1.35mg/ml, 20 min)
7. rinsed and washed with PBS(1x) (10 min, 10rpm)
8. rinsed with ddH20
9. dried with compressed air
10. incubated with Mowiol DABCO (5µl, overnight)

Fluorescence microscopy 16 (Nikon C2+ confocal microscope): GOPTS + B53

–> no spores detectable

Fluorescence microscopy 17 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP

–> GFP signal: GFP has been bound to nanobody

• 1-4) GOPTS + nanobody(Alexa647) + GFP
• 5) GOPTS + milk powder + GFP (negative control)
• 6) GOPTS + milk powder + NB (negative control)
• 7) GOPTS + GFP (positive control)
• 8) GOPTS (negative control)

Sample preparation 18: GOPTS + anti-GFP nanobody + GFP (spotting-mask)

1. incubation with anti-GFP nanobody ( 2µl/spot, 330µg/ml, 10 min, 4°C)
2. rinsing and blocking with milk powder(5%) (15 min, 10 rpm)
3. rinsing with PBS(1x)
4. incubated with GFP (2µl, 1.35mg/ml, 30 min, 4°C)
5. rinsed and washed with PBS(1x) (10 min, 10 rpm)
6. rinsed with ddH2O
7. (removed mask)
8. rinsed with ddH2O
9. dried with compressed air
10. incubated with Mowiol DABCO (overnight)

Fluorescence microscopy 18 (Nikon C2+ confocal microscope): GOPTS + antiGFP-nanobody + GFP (spotting-mask)

–> no GFP signal detectable

Sample preparation 19: GOPTS + anti-GFP nanobody + GFP (spotting-mask)

1. incubation with anti-GFP nanobody (2µl/spot, 330µg/ml, 15 min, 4°C)
2. rinsing with PBS(1x)
3. (removed mask)
4. rinsing and blocking with milk powder(5%) (15 min, 2 rpm)
5. (reattached mask)
6. incubated with GFP (2µl, 1.35mg/ml, 30 min, 4°C)
7. (removed mask)
8. rinsed and washed with PBS(1x) (10 min, 10 rpm)
9. rinsed with ddH2O
10. dried with compressed air
11. incubated with Mowiol DABCO (overnight)

Fluorescence microscopy 19 (Nikon C2+ confocal microscope): GOPTS + anti-GFP nanobody + GFP (spotting-mask)

–> slight GFP-signal on nanobody

Sample preparation 20: anti-GFP nanobody + B53 (in solution)

1. incubation of anti-GFP nanobody(Alexa647) (2µl, 165µg/ml, 15 min, 4°C) + B53
2. incubated on GOPTS-slide (2µl, 15 min)
3. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
4. rinsed and washed with PBS(1x) (2 x 1 min, 10 rpm)

–> problem: no washing away of unbound nanobody

Sample preparation 21: anti-GFP nanobody + B53 (in solution)

1. incubation with anti-GFP nanobody(Alexa647) (2µl, 165µg/ml, 30 min, 4°C) + B53
2. centrifuged (2 min, 13400 rpm)
3. washed with PBS(1x)
4. repeated steps 2 and 3
5. resuspended in PBS(1x) (30µl)
6. incubated on GOPTS-slide (2µl, 15 min)
7. rinsed and blocked with milkpowder(5%) (30 min, 10 rpm)
8. rinsed and washed with PBS(1x) (2 x 1 min, 10 rpm)

Sample preparation 22: GOPTS + GFP

1. incubation with GFP (1 µl, 1.35mg/ml, 30 min)
2. rinsed and blocked with milk powder(5%) (30 min)
3. rinsed and washed with PBS(1x) (10 min, 10 rpm)
4. rinsed with ddH2O
5. dried with compressed air
6. incubation with Mowiol DABCO (5µl, overnight)

–> Construct spores are ready for analysis

Fluorescence microscopy 22 (Nikon C2+ confocal microscope): GOPTS + GFP

–> spots with evenly distributed GFP

1. incubation with GFP (1 µl, 1.35mg/ml, 30 min)
2. spores centrifuged (2 min, 13,400 rpm)
3. washed with PBS(1x)
4. step 1 repeated
5. resuspended in PBS(1x)
6. incubation on GOPTS-slide(GFP) with spores (4µl, 400,000/µl, incubation time: see table below, 4°C)
7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
8. rinsed with PBS(1x)
9. rinsed with ddH2O
10. dried with compressed air
11. incubation with Mowiol DABCO (overnight)

Sample preparation 23: GOPTS + GFP + spores

–> no spores detectable

Sample preparation 24: GOPTS + GFP + spores (in EtOH resuspended)

1. incubation of GFP (4µl, 1,35g/ml) + XXX (4µl, 400,000/µl) (10 min, 4°C)
2. solution centrifuged (4 min, 13,400 rpm)
3. washed with PBS(1x)
4. step 2 repeated
5. resuspended in EtOH (250µl)
6. incubation on GOPTS-slide (4µl, 30 min, 4°C)
7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
8. rinsed with PBS(1x)
9. rinsed with ddH2O
10. dried with compressed air
11. incubation with Mowiol DABCO (overnight)

Fluorescence microscopy 24 (Nikon C2+ confocal microscope): GOPTS + GFP + spores (strains 3,6,7,9,wt) (in EtOH resuspended)

–> EtOH denaturated GFP: very weak signal. No spores detectable

Sample preparation 25: GFP + spores (strains 3,6,7,9) (in solution)

1. incubation of GFP (4µl, 1,35g/ml) + spores(strain 3,6,7,9) (4µl, 400,000/µl) (incubation time: see table below, 4°C)
2. centrifuged (2 min, 13400 rpm)
3. washed with PBS(1x)
4. repeated steps 2 and 3
5. resuspended in PBS(1x) (20µl)
6. incubated on GOPTS-slide (4µl, 10 min)
7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
8. rinsed and washed with PBS(1x) (10 min, 10 rpm)
9. rinsed with ddH2O
10. dried with compressed air
11. incubrnight)ation with Mowiol DABCO

Sample preparation 26: Incubation test: GFP + spores (in solution)

1. incubation of GFP (4µl, 1,35g/ml) + spores (4µl, 400,000/µl) (incubation time: see tables below, 4°C)
2. centrifuged (2 min, 13400 rpm)
3. washed with PBS(1x)
4. repeated steps 2 and 3)
5. resuspended in PBS(1x) (20µl))
6. incubated on GOPTS-slide (4µl, 10 min)
7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
8. rinsed and washed with PBS(1x) (10 min, 10 rpm)
9. rinsed with ddH2O
10. dried with compressed air
11. incubation with Mowiol DABCO (overnight)

Slide 1:

Slide 2:

Slide 3:

Sample preparation 28: GFP-volume test B: GFP + strain 7 (in solution, washed)

1. incubation of GFP (volume: see table below, 1,35g/ml) + strain 7 (4µl, 400,000/µl) (90 min, 4°C)
2. centrifuged (2 min, 13400 rpm)
3. washed with PBS(1x)
4. repeated steps 2 and 3)
5. resuspended in PBS(1x) (20µl)
6. incubated on GOPTS-slide (2µl, 90 min)
7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
8. rinsed and washed with PBS(1x) (10 min, 10 rpm)
9. rinsed with ddH2O
10. dried with compressed air
11. incubation with Mowiol DABCO (overnight)

Sample preparation 29: Spore-amount test: GFP + strain 7 (in solution)

1. incubation of GFP (0,5µl, 1,35g/ml) + strain 7 (amount: see table below, 400,000/µl) (90 min, 4°C)
2. centrifuged (2 min, 13400 rpm)
3. washed with PBS(1x)
4. repeated steps 2 and 3)
5. resuspended in PBS(1x) (20µl)
6. incubated on GOPTS-slide (2µl, 90 min)
7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
8. rinsed and washed with PBS(1x) (10 min, 10 rpm)
9. rinsed with ddH2O
10. dried with compressed air
11. incubation with Mowiol DABCO (overnight)

Fluorescence microscopy 24 (Nikon C2+ confocal microscope): GOPTS + GFP + spores (in EtOH resuspended)

–> spore concentration too high, too many layers of spores

Fluorescence microscopy 25 (Nikon C2+ confocal microscope): GFP + spores (in solution)

–> spore concentration too high, too many layers of spores

Fluorescence microscopy 26 (Nikon C2+ confocal microscope): Incubation test: GFP + spores (in solution)

–> spore concentration too high, too many layers of spores

Slide 1:

1.1) strain7 + GFP (30 min) (white field) 1.2) strain7 + GFP (30 min) (GFP-channel) 2.1) strain9 + GFP (30 min) (white field) 2.2) strain9 + GFP (30 min) (GFP-channel)







Slide 2:

1.1) strain 7 + GFP (90 min)(white field) 1.2) strain 7 + GFP (90 min)(GFP-channel) 2.1) strain WT + GFP (90 min)(white field) 2.2) strain WT + GFP (90 min)(GFP-channel)







Fluorescence microscopy 27 (Nikon C2+ confocal microscope): GFP-volume test A: GFP + strain 7 (in solution)

–> background fluorescence was too high

Fluorescence microscopy 28 (Nikon C2+ confocal microscope): GFP-volume test B: GFP + strain 7 (in solution)

–> background fluorescence was too high

Fluorescence microscopy 29 (Nikon C2+ confocal microscope): GFP + strain 7 (in solution)

–> spores and WT show similar levels of fluorescence, probably autofluorescence

1) strain 7 + GFP (white field) 2) strain 7 + GFP (GFP-channel) 3) WT + GFP (white field) 4) WT + GFP (GFP-channel)







Sample preparation 30: Wash-test A: GFP + spores (strains 3,6,7,9) (in solution)

1. incubation of GFP (1µl, 1,35g/ml) + spores (strain 3,6,7,9) (25,000.000 spores) (90 min, 4°C)
2. solution centrifuged (2 min, 13,400 rpm)
3. washed with PBS(1x)
4. step 2 and 3 repeated (2x/4x)
5. resuspended in EtOH (250µl)
6. incubation on GOPTS-slide (4µl, 30 min, 4°C)
7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
8. rinsed with PBS(1x)
9. rinsed with ddH2O
10. dried with compressed air
11. incubation with Mowiol DABCO (overnight)

Sample preparation 31: Wash-test B: GFP + spores (in solution)

1. incubation of GFP (1µl, 1,35g/ml) + spores (25,000.000 spores) (90 min, 4°C)
2. solution centrifuged (2 min, 13,400 rpm)
3. washed with TBST(0,005%)
4. steps 2 and 3 repeated (2x/4x)
5. resuspended in EtOH (250µl)
6. incubation on GOPTS-slide (4µl, 30 min, 4°C)
7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
8. rinsed with PBS(1x)
9. rinsed with ddH2O
10. dried with compressed air
11. incubation with Mowiol DABCO (overnight)

Fluorescence microscopy 30 (Nikon C2+ confocal microscope): Wash-test A: GFP + spores (strains 3,6,7,9) (in solution)

–> spore concentration too high, background fluorescence too high

Fluorescence microscopy 31 (Nikon C2+ confocal microscope): Wash-test B: GFP + spores (strains 3,6,7,9) (in solution)

–> indicates that strain 3 binds GFP

• 1.1) GFP + strain 3 (white field)
• 1.2) GFP + strain 3 (GFP-channel)
• 2.1) GFP + WT (negative control) (white field)
• 2.2) GFP + WT (negative control) (GFP-channel)
• 3) WT (negative control)

Sample preparation 32: Wash-test C: GFP + spores (in solution)

1. incubation of GFP (1µl, 1,35g/ml) + spores (25,000.000 spores) (90 min, 4°C)
2. solution centrifuged (2 min, 13,400 rpm)
3. washed with TBST(0,005%)
4. steps 2 and 3 repeated (1x/3x/5x)
5. resuspended in EtOH (250µl)
6. incubation on GOPTS-slide (4µl, 30 min, 4°C)
7. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
8. rinsed with PBS(1x)
9. rinsed with ddH2O
10. dried with compressed air
11. incubation with Mowiol DABCO (overnight)

Fluorescence microscopy 32 (Nikon C2+ confocal microscope): Wash-test B: GFP + spores (in solution)

–> spores and WT show similar levels of fluorescence, probably autofluorescence

Sample preparation 33: Blocking-test A: spores milk powder + GFP (in solution)

1. solution centrifuged (2 min, 13,400 rpm)
2. spores washed twice with PBS(1x) (2x 100µl)
3. spores resuspended and incubated in milk powder(5%) (100µl, incubation time: see table below)
4. incubation with GFP (5µl, 1,35g/ml, 90 min)
5. washed with PBS(1x) (3x 100µl)
6. resuspended in PBS(1x) (20 µl)
7. incubation on GOPTS-slide (4µl, 30 min, 4°C)
8. rinsed and blocked with milk powder(5%) (30 min, 10 rpm)
9. rinsed with PBS(1x)
10. rinsed with ddH2O
11. dried with compressed air
12. incubation with Mowiol DABCO (overnight)

Fluorescence microscopy 33 (Nikon C2+ confocal microscope): Blocking-test A: spores + milk powder + GFP (in solution)

–> spores and WT show similar levels of fluorescence

Sample preparation 34: Blocking-test B: spores + BSA + GFP (in solution)

1. solution centrifuged (2 min, 13,400 rpm)
2. spores (5,000,000) washed with PBS(1x) (3x 100µl)
3. spores resuspended and incubated in BSA(5%) (100µl, 60 min)
4. incubation with GFP (50µl, 100µg/ml, 60 min)
5. washed with TBST(0.05%) (5x 100µl)
6. resuspended in PBS(1x) (50 µl)
7. incubation with Mowiol DABCO (overnight)

Fluorescence microscopy 34 (Nikon C2+ confocal microscope): Blocking-test B: spores + BSA + GFP (in solution)

–> too little spores