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Latest revision as of 07:15, 20 November 2015

""

Labjournal Surchem September

Experiment 68a: Tetanus antigen on PDITC slides IX

2015.09.16

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

2015.09.16

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 pos. control (GFP) 0.5 mg/ml
5 BSA 5 mg/ml
6 Tetanus Antigen ~0.5 mg/ml
7 pos. control (GFP) 0.5 mg/ml
8 Tetanus Antigen ~0.5 mg/ml

* spots were incubated o/n at 4 °C

2015.09.17

Experiment/Protocol

Tet serum (-+): 024, 466

Reagent Step Flow rate (ul/min) Priming time Concentration
Buffer1603001x
BSA26060010 mg/ml
Buffer3606001x
Serum(-)4309001:10
Buffer5603001x
anti GFP6309001.5 µg/ml
Buffer7603001x
Serum(-)8309001:10
Buffer9603001x

Tet serum (+-): 208, 216

Reagent Step Flow rate (ul/min) Priming time Concentration
Buffer1603001x
BSA26060010 mg/ml
Buffer3606001x
Serum(-)4309001:10
Buffer5603001x
anti GFP6309001.5 µg/ml
Buffer7603001x
Serum(+)8309001:10
Buffer9603001x

GFP; Tet serum (-+): 287

Reagent Step Flow rate (ul/min) Priming time Concentration
Buffer1603001x
BSA26060010 mg/ml
Buffer3606001x
anti GFP4309001.5 µg/ml
Buffer5603001x
Serum(-)6309001:10
Buffer7603001x
Serum(+)8309001:10
Buffer9603001x

Results

Serum(-),anti GFP,Serum(+):

Slide 024: Quotion picture, flushed with 1:10 diluted blood serum, flush protocol: serum(-),anti GFP, serum(+)
Slide 024: binding curves

]

Slide 466: Quotion picture, flushed with 1:10 diluted blood serum, flush protocol: serum(-),anti GFP, serum(+)
Slide 466: binding curves

Serum(+),anti GFP,Serum(-):

Slide 208: Quotion picture, flushed with 1:10 diluted blood serum, flush protocol: serum(+),anti GFP, serum(-)
Slide 208: binding curves
Slide 216: Quotion picture, flushed with 1:10 diluted blood serum, flush protocol: serum(+),anti GFP, serum(-)
Slide 216: binding curves

anti GFP,Serum(-),Serum(+):

Slide 287: Quotion picture, flushed with 1:10 diluted blood serum, flush protocol: anti GFP, serum(-), serum(+)
Slide 287: binding curves

Experiment 67b: Tetanus antigen on PDITC slides VIII

2015.09.12

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

2015.09.13

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 - -
6 bBSA 200 µg/ml

* spots were incubated o/n at 4 °C

2015.09.14

Experiment/Protocol

Tet serum (-): 208, 254
Tet serum (+): 500, 024

Reagent Step Flow rate (ul/min) Priming time Concentration
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
anti-GFP4306001.5 ug/ml
Buffer5603001x
Serum SIG002 (-)/(+)6309001:20
Buffer7603001x
Anti Human8306005 ug/ml
Buffer9603001x
StrepCy510306005 ug/ml
Buffer11603001x

Results

Blood Serum(-):

Slide 208: Quotion picture, flushed with 1:20 diluted blood serum (-)
Slide 208: binding curves

]

Slide 254: Quotion picture, flushed with 1:20 diluted blood serum (-)
Slide 254: binding curves

Blood serum(+):

Slide 024: Quotion picture, flushed with 1:20 diluted blood serum (+)
Slide 024: binding curves
Slide 500: Quotion picture, flushed with 1:20 diluted blood serum (+)
Slide 500: binding curves

Experiment 67a: Tetanus antigen on PDITC slides VII

2015.09.12

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

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

* spots were incubated o/n at 4 °C

2015.09.13

Experiment/Protocol

Tet serum (-): 443, 466
Tet serum (+): 423, 424

Reagent Step Flow rate (ul/min) Priming time Concentration
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
anti-GFP4306001.5 ug/ml
Buffer5603001x
Serum SIG002 (-)/(+)6309001:10
Buffer7603001x
Anti Human8306005 ug/ml
Buffer9603001x
StrepCy510306005 ug/ml
Buffer11603001x

Results

Blood Serum(-):

Slide 443: Quotion picture, flushed with 1:10 diluted blood serum (-)
Slide 443: binding curves

]

Slide 466: Quotion picture, flushed with 1:10 diluted blood serum (-)
Slide 466: binding curves

Blood serum(+):

Slide 423: Quotion picture, flushed with 1:10 diluted blood serum (+)
Slide 423: binding curves
Slide 424: Quotion picture, flushed with 1:10 diluted blood serum (+)
Slide 424: binding curves

Experiment 66b: PDITC slides for iRIf

2015.09.06

Experiment/Protocol

  • 6 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

Experiment 66a: PDITC slides for iRIf

2015.09.06

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

2015.09.10

Experiment/Protocol

  • slides were sandwiched with NTA and incubated at 4°C o/n

2015.09.10

Experiment/Protocol

  • slides were blocked with APTES blocking solution for 1 h
  • slides were washed 2x with PBS
  • slides were incubated with NiSO4-solution for 1 h and 20 min
  • slides were washed with PBS/2x diluted PBS

uploaded 20150906

Experiment 65c: Tetanus antigen PDITC slides for iRIf VI

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: 30 min
  • stored at 4 °C

2015.09.05

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 - -
6 bBSA 200 µg/ml

2015.09.06

Experiment/Protocol

Flush protocol:

Tet serum (-): 443
Tet serum (+): 466

Reagent Step Flow rate (ul/min) Priming time Concentration
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
anti-GFP4306001.5 ug/ml
Buffer5603001x
Serum SIG002 (-)/(+)6306001:4
Buffer7603001x
StrepCy58306005 ug/ml
Buffer9603001x

Tet serum (-): 208
Tet serum (+): 303

Reagent Step Flow rate (ul/min) Priming time Concentration
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
anti-GFP4306001.5 ug/ml
Buffer5603001x
Serum SIG002 (-)/(+)6606001:6
Buffer7603001x
StrepCy58306005 ug/ml
Buffer9603001x

Note: the amount of flushed serum

Results

Blood Serum(-):

Slide 208: Quotion picture, flushed with 1:6 diluted blood serum (-)
Slide 208: binding curves

]

Slide 443: Quotion picture, flushed with 1:4 diluted blood serum (-)
Slide 443: binding curves

Blood serum(+):

Slide 303: Quotion picture, flushed with 1:6 diluted blood serum (+)
Slide 303: binding curves
Slide 466: Quotion picture, flushed with 1:4 diluted blood serum (+)
Slide 466: binding curves

→ for the blood serum after immunization (+) the iRIf signal for tetanus binding is higher than for the blood serum before immunization (-)

→ there are differences to the results of experiment 59a, the overall tetanus signal is much weaker as well as the binding of the blood serum to bBSA

Experiment 65b: Tetanus antigen PDITC slides for iRIf V

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: 30 min

2015.09.04

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 - -
6 bBSA 200 µg/ml

2015.09.05

Experiment/Protocol

Flush protocol:

SIG 003 (+): 012(no serum was flushed over the slide), 315, 426
SIG 002 (+): 215

Reagent Step Flow rate (ul/min) Priming time Concentration
Buffer1606001x
BSA26060010 mg/ml
Buffer3606001x
anti-GFP4306001.5 ug/ml
Buffer5603001x
Blood serum6306001:30
Buffer7603001x
StrepCy58306005 ug/ml
Buffer9603001x

Results

SIG 003:

Slide 012: Quotion picture
Slide 012: binding curves
Slide 315: Quotion picture
Slide 426: Quotion picture
Slide 426: binding curves

→ slide 012 was not flushed with any blood serum → for blood serum SIG 003 only a slight antigen/antibody binding is detectable (slide 426)


SIG 002:

Slide 215: Quotion picture
Slide 215: binding curves

→ slight antigen/antibody binding is detectable

→ no anti GFP/ GFP binding detected

→ only weak binding of the blood serum to bBSA → difference to results of experiment 59a

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

2015.08.31

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)

2015.09.04

Experiment/Protocol

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

Results

PDITC:

Slide 216: Quotion picture
Slide 216: binding curves
Slide 254: Quotion picture
Slide 254: binding curves

Ni-NTA:

Slide 102: Quotion picture
Slide 102: binding curves
Slide 502: Quotion picture
Slide 502: binding curves

→ the his tagged, cell free GFP was specificly bound by the Ni NTA surface, whereas there was no detectable amount of cell free GFP bound on the PDITC surfaces

→ the PDITC surface binds all proteins in the spotted cell free expression mix eaqually good, whereas the Ni-NTA surface specificly binds only the his tagged, cell free expressed GFP

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