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Labjournal Surchem September

Experiment 68a: Tetanus antigen on PDITC slides IX

• protocols used :Plasma activation, iRIf slide preparation

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
Buffer	1	60	300	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
Serum(-)	4	30	900	1:10
Buffer	5	60	300	1x
anti GFP	6	30	900	1.5 µg/ml
Buffer	7	60	300	1x
Serum(-)	8	30	900	1:10
Buffer	9	60	300	1x

Tet serum (+-): 208, 216

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	300	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
Serum(-)	4	30	900	1:10
Buffer	5	60	300	1x
anti GFP	6	30	900	1.5 µg/ml
Buffer	7	60	300	1x
Serum(+)	8	30	900	1:10
Buffer	9	60	300	1x

GFP; Tet serum (-+): 287

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	300	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	600	1x
anti GFP	4	30	900	1.5 µg/ml
Buffer	5	60	300	1x
Serum(-)	6	30	900	1:10
Buffer	7	60	300	1x
Serum(+)	8	30	900	1:10
Buffer	9	60	300	1x

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

• protocols used :Plasma activation, iRIf slide preparation

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
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	900	1:20
Buffer	7	60	300	1x
Anti Human	8	30	600	5 ug/ml
Buffer	9	60	300	1x
StrepCy5	10	30	600	5 ug/ml
Buffer	11	60	300	1x

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

• protocols used :Plasma activation, iRIf slide preparation

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
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	900	1:10
Buffer	7	60	300	1x
Anti Human	8	30	600	5 ug/ml
Buffer	9	60	300	1x
StrepCy5	10	30	600	5 ug/ml
Buffer	11	60	300	1x

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

• protocols used :Plasma activation, iRIf slide preparation

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

• protocols used :Plasma activation, iRIf slide preparation

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

• 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: 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
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	1:4
Buffer	7	60	300	1x
StrepCy5	8	30	600	5 ug/ml
Buffer	9	60	300	1x

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

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	60	600	1:6
Buffer	7	60	300	1x
StrepCy5	8	30	600	5 ug/ml
Buffer	9	60	300	1x

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

• 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: 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
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
Blood serum	6	30	600	1:30
Buffer	7	60	300	1x
StrepCy5	8	30	600	5 ug/ml
Buffer	9	60	300	1x

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

• protocols used :Plasma activation, iRIf slide preparation

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
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

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

• 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(-):

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

• 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(-):

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

• 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.

Slide 208: ROI selection

Slide 208: Binding curves