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Latest revision as of 07:14, 20 November 2015

""

Labjournal iRIf September

Measuring own blood (tetanus vaccination)

16.09.2015 (spotted)

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

17.09.2015 (measured)

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

Measuring : The **DIA Chip**

14.09.2015 (stamped)

PDMS Dia-Chip stamp (incubated 30 minutes with 1 mg/ml GFP) was stamped onto plasmaactivated slide. Kept in 10 mg/ml BSA in PBS at 4° until measurement.

16.09.2015 (measured)

Slides were previously blocked with the backside of a PDMS flowcell to avoid airbubble issues due to the hydrophobicity of the stamp.

Reagent Step Flow rate (ul/min) Priming time Concentration
Buffer1603001x
BSA26060010 mg/ml
Buffer3606001x
anti-GFP4306005 ug/ml
Buffer5603001x
StrepCy56603005 ug/ml
Buffer9603001x
Slide 287: Cy5 signal after iRIf measurement detected in microarray scanner
Slide 303: Quotient picture of the anti-GFP step

Rabbit-serum (GFP immunized/not immunized) on PDITC

14.09.2015 (spotted)

Spotting pattern:

# spot Concentration
1 pos. control (bBSA) 0.2 mg/ml
2 neg. control (BSA) 0.2 mg/ml
3 His-GFP desalt 1mg/ml
4 pos. control (bBSA) 0.2 mg/ml
5 neg. control (BSA) 0.2 mg/ml
6 His-GFP desalt 1mg/ml
7 His-mCherry desalt 1 mg/ml
8 His-mCherry desalt 1 mg/ml

15.09.2015 (measured)

Flush protocol: Positive serum (504,424,466). Negative serum (423).

Reagent Step Flow rate (ul/min) Priming time Concentration
Buffer1603001x
BSA26060010 mg/ml
Buffer3606001x
StrepCy54306005 ug/ml
Buffer5603001x
Serum (+)/(-)8309001:30
Buffer9606001x
Anti-Rabbit10603005 ug/ml
Buffer11603001x

*Slide 466 was measured with 10 ug/ml Anti-Rabbit

Results: Serum (-) fromm rabbit that was not immunized to GFP shows no binding to the GFP spot.
Serum (+) from rabbit that was immunized to GFP shows extensive binding to the GFP spot.

Slide 423 (serum(-)): Selections of ROIs
Slide 423 (serum(-)): Binding curves

Slide 424 (serum(+)): Selection of ROIs
Slide 424 (serum(+)): Binding curves

Slide 466 (serum(+)): Selections of ROIs

Measuring own blood (Tetanus vaccination)

13.09.2015 (spotted)

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

14.09.2015 (measured)

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

Measuring own blood serum (Tetanus vaccination)

12.09.2015 (spotted)

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

13.09.2015 (measured)

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

Measuring own blood (Tetanus vaccination)

05.09.2015 (spotted)

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

06.09.2015 (measured)

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

Measuring own blood: Tetanus (-), Tetanus (+)

04.09.15 (spotted)

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

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 serum6306001:10/1:30
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

Specific surface (Ni-NTA) vs. unspecific surface (PDITC): cellfree expressed GFP

04.09.2015 (spotted)

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

05.09.2015 (measured)

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

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

Measuring own blood: Tetanus (-), Tetanus (+) Sig002

02.09.2015 (spotted) 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

03.09.2015 (measured)

Tet (-): 287,429 (1:30 diluted serum) Tet (+): 443, 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
Slide 287: ROI selection, ROIs were chosen in similar manner for the other slides
Slide 287: Binding curves; serum (-) diluted 1:10
Slide 443: Binding curves; serum (+) diluted 1:10
Slide 429: Binding curves; serum (-) diluted 1:30
Slide 423: Binding curves, serum (+) diluted 1:30

Distinguish between different antigen/antibody interaction (Salmonella) (on PDITC) [finished]

01.09.2015 (spotted)

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 last slide (208) changed protocol: only 1 ug/ml anti-GFP used; anti-Salmonella desalt was mixed 1:1 with anti-Salmonella lysate

02.09.2015 (measured)

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

Note: Background of Slides 208 looked nicest.

Slide 208: ROI selection
Slide 208: Binding curves
Slide 215: Binding curves
Slide 216: Binding curves