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Revision as of 08:11, 10 September 2015

""

Labjournal Surchem September

Experiment 65b: PDITC slides for iRIf

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

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
anti-GFP4306001.5 ug/ml
Buffer5603001x
Serum SIG002 (-)/(+)6306001:10/1:30
Buffer7603001x
StrepCy58306005 ug/ml
Buffer9603001x

Experiment 65a: cell free GFP on NiNTA and PDITC slides for iRIf

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
anti-GFP4306001.5 ug/ml
Buffer5603001x
StrepCy58306005 ug/ml
Buffer9603001x

Experiment 64c: Tetanus antigen PDITC slides for iRIf IV

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
anti-GFP4306001.5 ug/ml
Buffer5603001x
Serum SIG002 (-)/(+)6306001:10/1:30
Buffer7603001x
StrepCy58306005 ug/ml
Buffer9603001x

Results

Serum(-):

Slide 500: Quotion picture, Tet serum (-), 1:10 diluted
Slide 500: binding curves

Serum(+):

Slide 024: Quotion picture, Tet serum (+), 1:10 diluted

→ bad measurement, no binding curves were obtained

Slide 504: Quotion picture, Tet serum (+), 1:30 diluted
Slide 504: binding curves

→ 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

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
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
anti-GFP4306001.5 ug/ml
Buffer5603001x
Serum SIG002 (-)/(+)6209001:10
Buffer7603001x
StrepCy58306005 ug/ml
Buffer9603001x

Results

Serum(-):

Slide 287: Quotion picture, Tet serum (-), 1:10 diluted
Slide 287: binding curves
Slide 429: Quotion picture, Tet serum (-), 1:30 diluted
Slide 429: binding curves

→ 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(+):

Slide 443: Quotion picture, Tet serum (+), 1:10 diluted
Slide 443: binding curves
Slide 423: Quotion picture, Tet serum (+), 1:30 diluted
Slide 423: binding curves

→ 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

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)1606001x
BSA26060010 mg/ml
Buffer (TBS)3606001x
anti-GFP4306001,5 ug/ml
Buffer (TBS)5603001x
anti-Salmonella (13, desalt)630900undiluted
Buffer (TBS)7603001x
StrepCy58306005 ug/ml
Buffer (TBS)9603001x

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.

Slide 208: ROI selection
Slide 208: Binding curves