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Revision as of 10:03, 9 September 2015
Labjournal Surchem September
Experiment 65b: PDITC slides for iRIf
- protocols used :Plasma activation, iRIf slide preparation
2015.09.04
Experiment/Protocol
- 4 PDITC slides were prepared
- Plasma activation: 40 L/h, 5 min
- APTES incubation: 30 min
- PDITC incubation: 2 h
- desiccator: 30 min
- stored at 4 °C
Experiment 65b: Tetanus antigen PDITC slides for iRIf V
- protocols used :Plasma activation, iRIf slide preparation
2015.09.04
Experiment/Protocol
- 4 PDITC slides were prepared
- Plasma activation: 40 L/h, 5 min
- APTES incubation: 30 min
- PDITC incubation: 2 h
- desiccator: 30 min
Spotting pattern: 
| # | spot | Concentration |
|---|---|---|
| 1 | pos. control (GFP) | 0.5 mg/ml |
| 2 | BSA | 5 mg/ml |
| 3 | Tetanus Antigen | ~0.5 mg/ml |
| 4 | - | - |
| 5 | - | - |
| 6 | bBSA | 200 µg/ml |
2015.09.05
Experiment/Protocol
Flush protocol:
Tet serum (-):
Tet serum (+):
| Reagent | Step | Flow rate (ul/min) | Priming time | Concentration |
|---|---|---|---|---|
| Buffer | 1 | 60 | 600 | 1x |
| BSA | 2 | 60 | 600 | 10 mg/ml |
| Buffer | 3 | 60 | 600 | 1x |
| anti-GFP | 4 | 30 | 600 | 1.5 ug/ml |
| Buffer | 5 | 60 | 300 | 1x |
| Serum SIG002 (-)/(+) | 6 | 30 | 600 | 1:10/1:30 |
| Buffer | 7 | 60 | 300 | 1x |
| StrepCy5 | 8 | 30 | 600 | 5 ug/ml |
| Buffer | 9 | 60 | 300 | 1x |
Experiment 65a: cell free GFP on NiNTA and PDITC slides for iRIf
- protocols used :Plasma activation, iRIf slide preparation
2015.09.04
Experiment/Protocol
- 2 PDITC slides were prepared (216,254)
- Plasma activation: 40 L/h, 5 min
- APTES incubation: 30 min
- PDITC incubation: 2 h
- desiccator: 30 min
- 2 Ni NTA slides from experiment 63b were also spotted (102,502)
Spotting pattern:
| # | spot | Concentration |
|---|---|---|
| 1 | pos. control (GFP) | 0.5 mg/ml |
| 2 | cell free expression mix without DNA (KK) | |
| 3 | cell free expressed HA-GFP-His6-His6 (KK) | |
| 4 | pos. control (GFP) | 0.5 mg/ml |
| 5 | cell free expression mix without DNA (KK) | |
| 6 | cell free expressed His10-GFP-Spy (KK) | |
| 7 | BSA | 5 mg/ml |
| 8 | bBSA | 200 µg/ml |
2015.09.05
Experiment/Protocol
Flush protocol:
| Reagent | Step | Flow rate (ul/min) | Priming time | Concentration |
|---|---|---|---|---|
| Buffer | 1 | 60 | 600 | 1x |
| BSA | 2 | 60 | 600 | 10 mg/ml |
| Buffer | 3 | 60 | 600 | 1x |
| anti-GFP | 4 | 30 | 600 | 1.5 ug/ml |
| Buffer | 5 | 60 | 300 | 1x |
| StrepCy5 | 8 | 30 | 600 | 5 ug/ml |
| Buffer | 9 | 60 | 300 | 1x |
Experiment 64c: Tetanus antigen PDITC slides for iRIf IV
- protocols used :Plasma activation, iRIf slide preparation
2015.08.31
Experiment/Protocol
- 4 PDITC slides were prepared
- Plasma activation: 40 L/h, 5 min
- APTES incubation: 30 min
- PDITC incubation: 2 h
- desiccator: 15 min
- stored at 4 °C
2015.09.03
Experiment/Protocol
Spotting pattern:
| # | spot | Concentration |
|---|---|---|
| 1 | pos. control (GFP) | 0.5 mg/ml |
| 2 | BSA | 5 mg/ml |
| 3 | Tetanus Antigen | ~0.5 mg/ml |
| 4 | - | - |
| 5 | mCherry | 0.5 mg/ml |
| 6 | bBSA | 200 µg/ml |
2015.09.04
Experiment/Protocol
Flush protocol:
Tet serum (-): 500(1:10 diluted serum)
Tet serum (+): 024 (1:10 diluted serum), 504 (1:30 diluted serum)
| Reagent | Step | Flow rate (ul/min) | Priming time | Concentration |
|---|---|---|---|---|
| Buffer | 1 | 60 | 600 | 1x |
| BSA | 2 | 60 | 600 | 10 mg/ml |
| Buffer | 3 | 60 | 600 | 1x |
| anti-GFP | 4 | 30 | 600 | 1.5 ug/ml |
| Buffer | 5 | 60 | 300 | 1x |
| Serum SIG002 (-)/(+) | 6 | 30 | 600 | 1:10/1:30 |
| Buffer | 7 | 60 | 300 | 1x |
| StrepCy5 | 8 | 30 | 600 | 5 ug/ml |
| Buffer | 9 | 60 | 300 | 1x |
Results
Serum(-):
Serum(+):
→ bad measurement, no binding curves were obtained
→ signal for 1:10 dilution of the blood serum much better → continue with 1:10 dilution
Experiment 64b: Tetanus antigen PDITC slides for iRIf III
- protocols used :Plasma activation, iRIf slide preparation
2015.08.31
Experiment/Protocol
- 4 PDITC slides were prepared
- Plasma activation: 40 L/h, 5 min
- APTES incubation: 30 min
- PDITC incubation: 2 h
- desiccator: 15 min
- stored at 4 °C
2015.09.02
Experiment/Protocol
Spotting pattern:
| # | spot | Concentration |
|---|---|---|
| 1 | pos. control (GFP) | 0.5 mg/ml |
| 2 | mCherry | 0.5 mg/ml |
| 3 | Tetanus Antigen | ~0.5 mg/ml |
| 4 | BSA | 10 mg/ml |
| 5 | BSA +bBSA | 10 mg/ml |
| 6 | bBSA | 200 µg/ml |
note: bBSA on spot 5 was incubated for only a minute and then replaced with BSA
2015.09.03
Experiment/Protocol
Flush protocol:
Tet serum (-): 287 (1:10 diluted serum),429 (1:30 diluted serum)
Tet serum (+): 443 (1:10 diluted serum), 423 (1:30 diluted serum)
| Reagent | Step | Flow rate (ul/min) | Priming time | Concentration |
|---|---|---|---|---|
| Buffer | 1 | 60 | 600 | 1x |
| BSA | 2 | 60 | 600 | 10 mg/ml |
| Buffer | 3 | 60 | 600 | 1x |
| anti-GFP | 4 | 30 | 600 | 1.5 ug/ml |
| Buffer | 5 | 60 | 300 | 1x |
| Serum SIG002 (-)/(+) | 6 | 20 | 900 | 1:10 |
| Buffer | 7 | 60 | 300 | 1x |
| StrepCy5 | 8 | 30 | 600 | 5 ug/ml |
| Buffer | 9 | 60 | 300 | 1x |
Results
Serum(-):
→ higher dilution of the blood serum leads to weaker iRif signal for mCherry, BSA,BSA+bBSA and bBSA but also for the tetanus antigen
Serum(+):
→ higher dilution of the blood serum leads to weaker iRif signal for mCherry, BSA,BSA+bBSA and bBSA but also for the tetanus antigen
→ signal of the tetanus antigen spot very weak compared to experiment 59a → use fresh stock of tetanus antigen next time
→ unspecific binding to the BSA spot is most of the time weaker than to mCherry → from now on BSA will be used as negative contol for blood serum
Experiment 64a: Salmonella antigen on PDITC slides for iRIf II
- protocols used :Plasma activation, iRIf slide preparation
2015.08.31
Experiment/Protocol
- 3 PDITC slides were prepared
- Plasma activation: 40 L/h, 5 min
- APTES incubation: 30 min
- PDITC incubation: 2 h
- desiccator: 15 min
- stored at 4 °C
2015.09.01
Experiment/Protocol
Spotting pattern: 
| # | spot | Concentration |
|---|---|---|
| 1 | pos. control (GFP) | 0.5 mg/ml |
| 2 | bBSA | 0.5 mg/ml |
| 3 | Salmonella Antigen (15) | undiluted |
Slides (3): 208,215,216
2015.09.02
Experiment/Protocol
Flush protocol:
| Reagent | Step | Flow rate (ul/min) | Priming time | Concentration |
|---|---|---|---|---|
| Buffer (TBS) | 1 | 60 | 600 | 1x |
| BSA | 2 | 60 | 600 | 10 mg/ml |
| Buffer (TBS) | 3 | 60 | 600 | 1x |
| anti-GFP | 4 | 30 | 600 | 1,5 ug/ml |
| Buffer (TBS) | 5 | 60 | 300 | 1x |
| anti-Salmonella (13, desalt) | 6 | 30 | 900 | undiluted |
| Buffer (TBS) | 7 | 60 | 300 | 1x |
| StrepCy5 | 8 | 30 | 600 | 5 ug/ml |
| Buffer (TBS) | 9 | 60 | 300 | 1x |
last slide (208) changed protocol: only 1 ug/ml anti-GFP used; anti-Salmonella desalt was mixed 1:1 with anti-Salmonella lysate
Results
Note: Slide 208 had the best signal.
