Team:Freiburg/Labjournals/SurChem/March
Labjournal Surchem March
Experiment 3 : APTES/PDITC slides
2015.03.31
| iRIF-slides: | BW-A-01-001-[314] |
| BW-A-01-002-[315] | |
| normale Detektion (strep-cy5): | SN-A-01-001 |
| SN-A-01-002 | |
| SN-A-01-003 | |
| SN-A-01-004 | |
| Coomassie-Detektion: | SB-A-08-001 |
| SB-A-08-002 | |
| SB-A-08-003 | |
| SB-A-08-004 |
Experimental/Protocol:
Protocols for APTES/PDITC-slides and plasma activation (quantities x2 because 10 slides)
- For SB-A-08-001 and SB-A-08-002 BSA was directly added on the PDITC surface according to the protocol (–> APTES+PDITC+BSA). Slides were then incubated in Coomassie for 30 min and cleaned with water. Slides SB-A-08-003 and SB-A-08-004 were directly incubated in Coomassie for 30 min and cleaned with water. No BSA was added.
2015.04.01
Experimental/Protocol:
- SN-A-01-001 and SN-A-01-004 were coated/overlayed with 40 µL of Strep-Cy5
- SN-A-01-002 and SN-A-01-003 formed a Strep-Sandwich (80 µL Strep-Cy5)
- BW-A-01-001 and BW-A-01-002 were prepared according to the protocol up to the blocking with BSA. Strep-Cy5 was added in the flow cell during iRIF measurement.
Results:
| slide | results |
|---|---|
| BW-A-01-001-[314] | All 5 spots could be detected but the bBSA-spots had a strong comet tail. This might be due to proteins adhering to the surface during the washing step. The intensity of the bBSA-spots was about 20 000. DNA-spots had an intensity of 2800-4000. The background was about 100. |
| BW-A-01-002-[315] | |
| SN-A-01-001 | |
| SN-A-01-002 | |
| SN-A-01-003 | |
| SN-A-01-004 | |
| SB-A-08-001 | |
| SB-A-08-002 | |
| SB-A-08-003 | |
| SB-A-08-004 |
Remarks
- After incubation in Strep-Cy5 slides should not be rinsed with water but let slip into a „Kegelvase“ filled with water to prevent comet tails.
Questions and Tasks
Experiment 2: Introduction to APTES/PDITC-surface chemistry I
2015.03.25
SB-01
SB-02
SB-03
SB-04
SB-05
Experimental/Protocol:
Protocols for APTES/PDITC and Plasma activation
APTES-solution for 5 slides (V = 20 mL)
| acetone | 18.8 mL |
| aqua dest. | 1 mL |
| APTES | 0.2 mL |
PDITC-solution for 5 slides (V = XX mL)
| Pyridine | 2.0 mL |
| DMF | 18.0 mL |
| PDITC | 40 mg |
DNA-control-solution for 5 slides (10 spots á 0.5 µL, 2 µM)
| NaPi-buffer pH 8.3 | 15 µL |
| aqua RNAse free | 9 µL |
| DNA | 6 µL |
- On each slide : 2 spots DNA and 3 spots bBSA
- Incubate overnight
2015.03.26
Experimental/Protocol:
- Spots on slides SB-01 and SB-03 were dried on air.
- Spots on slides SB-02 SB-04 and SB-05 were rinsed off with water.
- For blocking with BSA the Sandwich-technique was used. Slide SB-02 was just ovrlayed with 70 µL BSA (no sandwich).
Results:
| slide | results |
|---|---|
| SB-01 | All 5 spots could be detected but slide has not been washed carefully enough. |
| SB-02 | Not beautiful. |
| SB-03 | 2 spots observed |
| SB-04 | All 5 spots observed + 2 more above the expected pattern. Spots must have been transferred from one slide to the other during sandwich-time. |
| SB-05 | 5 spots observed. All the bBSA spots had diverged. |
Remarks
- Always store APTES with Parafilm
- Slides with APTES+PDITC can be stored up to 2 or 3 days
- Spot-technique is used on PDITC-slides because the reaction is slower and homogene (avoids comet tails)
Questions and Tasks
Experiment 1: Introduction to GOPTS-surface chemistry I
2015.03.24
JB-01
NW-01
JE-01
SB-01
LP-01
XYZ-01 (Günther)
Experimental/Protocol:
Protocols for GOPTS surface, Plasma activation and Protein stamping with PDMS
- bBSA-solution (50-60 µL) was pipetted on stamps
- After stamping on slides, both stamps and slides were stored at 4°C
2015.03.25
Experimental/Protocol:
- Slides from fridge were sandwiched with 80 µL BSA-solution in PBS-buffer (10 mg/mL)
- Slides were incubated for 30 min
- After separation slides were cleaned with aqua dest.
- Dried with wafergun
- Slide-sandwiches with 80 µL Streptavidin-Cy5-solution (0.5 µg/mL) per sandwich
- Incubated in petri dish for 30 min with tissue over petri dish
- After separtion slides were put into aqua dest.
- Dried with wafergun
- Measured with fluorescence scanner
Results:
| slide | results |
|---|---|
| JB-01 | |
| NW-01 | |
| JE-01 | |
| SB-01 | |
| LP-01 | |
| XYZ-01 |
Remarks
- When incubating the stamps with protein solution, immediately restore unused stamp material in order to protect it from dust!
- When stamping do not leave the stamp longer than 5-10 s (maximum 30 s) on surface.
- Weak intensities of fluorescence might be due to slow preparation of slides.
Questions and Tasks
- Be faster during cleaning processes!
