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iRIf Labjournal August

Distinguish between different antigen/antibody interaction (GFP, mouse, rabbit) (on PDITC) [finished]

31.08.2015 (spotted) spotting pattern:

slides: 424, 012

#	spot	Concentration
1	GFP	0.5 mg/ml
2	anti HCV (rabbit)	0.5 mg/ml
3	anti Tetanus (mouse)	0.5 mg/ml

slide: 254

#	spot	Concentration
1	GFP	1 mg/ml
2	anti HCV (rabbit)	1 mg/ml
3	anti Tetanus (mouse)	1 mg/ml

01.09.2015 (measured)

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	3 ug/ml
Buffer	5	60	300	1x
anti- rabbit	6	30	600
Buffer	7	60	300	1x
anti- mouse	8	30	600
Buffer	9	60	300	1x

Slide 12: ROI selection, ROIs were selected in similar manner for the other slides

Slide 12: Binding curves

Slide 257: Binding curves

Slide 424: Binding curves

Affibody (Stockholm cooporation)

30.08.2015 (spotted)

spotting pattern:

slide-#:315

#	spot	Concentration
1-3	rhErbB2/Fc	100 µg/ml
4	GFP (desalt)	1 mg/ml
5	Tetanus antigene	100 µg/ml

31.08.2015 (measured)

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
Affibody	6	20	900	1:3 diluted
Buffer	7	60	300	1x
Affibody	8	20	900	undiluted
Buffer	9	60	300	1x
Anti-His	10	30	600	20 ug/ml
Buffer	11	60	300	1x

As can be seen in the evaluation of the binding curves (slide 315), the affibody did not bind to the antigen or it is too small to be detected with iRIf. Anti-His confirmed antigen presence on the slides.

Slide 315: ROI selection

Slide 315: Binding curves

Measuring own blood (Tet (-) / Tet (+)) on PDITC

30.08.2015 (spotted) Slides: 2 slides, 303, 466 Tet (-): 303, 466

Spotting pattern:

#	spot	Concentration
1	pos. control (GFP)	0.5 mg/ml
2	neg. con (Salmonella)	(1.24 mg/ml) desalt 1:3 diluted
3	Tetanus	(0.46 mg/ml) desalt undiluted

31.08.2015 (measured)

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
Serum SIG002 (-)	6	20	900	1:10 (466) 1:20 (303)
Buffer	7	60	300	1x

Slide 466: Roi selection

Slide 466: Binding curves

Slide 303: Binding curves

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

29.08.2015 (spotted)

Spotting pattern:

#	spot	Concentration
1	pos. control (GFP)	1 mg/ml
2	neg. control (mCherry)	1mg/ml
3	Salmonella Antigen (15)	undiluted

Slides: 208, 216, 423, 500

208: with PBS 423: with TBS 216: with TBS 500: with TBS (changed flush protocol: 1 ug/ml a-GFP; and used anti-Salmonella lysate 1:2 diluted in 5 mg/ml BSA in TBS)

30.08.2015 (measured)

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	3 ug/ml
Buffer	5	60	300	1x
anti-Salmonella (13, desalt)	6	30	900	1:2 Dilution in 5 mg/ml BSA in PBS
Buffer	7	60	300	1x

Slide 216: ROI selection; ROI selection was done in similar manner for the other slides

Slide 216: Binding curves

Slide 208: Binding curves

Slide 423: Binding curves

Slide 500: Binding curves

Measuring own blood (Tetanus unimmunized vs. immunized) on PDITC

29.08.2015 (spotted) Slides: 24, 287, 426, 443

Spotting pattern:

#	spot	Concentration
1	pos. control (GFP)	1 mg/ml
2	neg. con (Salmonella)	desalt 1:3
3	Tetanus	desalt undiluted

30.08.2015 (measured)

Slides: 287, 24 (with Serum (-)) #287: Serum (-) was diluted only 1:10; changed to 1:30 because Serum(-) quite high Signal at negative control and Tetanus spot 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
Serum (-) Sig002	6	30	900	1:30 diluted in 5 mg/ml BSA in PBS
Buffer	7	60	300	1x

Slides: 426,443 (with Serum (+)) 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
Serum (+) Sig002	6	30	900	1:30 diluted in 5 mg/ml BSA in PBS
Buffer	7	60	300	1x

Note: Slide 426 looked nicest

Slide 287: ROI selection; ROIs of the other slides were chosen in similar manner

Slide 24: Binding curves

Slide 287: Binding curves

Slide 426: Binding curves

Slide 443: Binding curves

Measuring on-slide cellfree expressed GFP (Kokos Mix, AG Roth Mix, Reticolozytes Mix)

#254: retikulozyte #12: roth #423: koko

27.08.2015 (spotted) Check out surface chemistry: Experiment 61: DNA on PDMS 6.0 for previous treatment

28.08.2015 (measured)

Flush Protocol:

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	450	1x
BSA	2	60	450	10 mg/ml
Buffer	3	60	450	1x
anti-GFP	4	30	600	3 ug/ml
Buffer	5	60	300	1x
strep-cy5	6	60	300	5 ug/ml
Buffer	7	60	300	1x

Plateready after iRIf showed cy5 signal at some spots on every slide. This means (probably) that the biotinylated a-GFP bound to GFP. Looking at the cy5 spots, analogous areas in RIfs were taken for spot evaluation. Spot evaluation showed anti-GFP binding (but less signal) to GFP in every case. → On slide expression of GFP at least seemed to work to a little amount

Slide 12 (expressed with AG Roth mix): Platereader cy5 signal after iRIf

Slide 12 (expressed with AG Roth mix): ROI selection

Slide 12 (expressed with AG Roth mix): Binding curves

Slide 254 (expressed with retikulozyte mix): Platereader cy5 signal after iRIf

Slide 254 (expressed with retikulozyte mix): ROI selection

Slide 254 (expressed with retikulozyte mix): Binding curves

Slide 423 (expressed with Koko mix): Platereader cy5 signal after iRIf

Slide 423 (expressed with Koko mix): ROI selection

423 (expressed with Koko mix): Binding curves

Measuring on-slide cellfree expressed GFP

25.08.2015 (spotted) spotting pattern:

slide-#: 429, 424

#	spot	Concentration
1-3	MM + DNA
4-5	MM - DNA)
6	GFP (off-slide cell free expressed)
7	His-GFP desalt.	~1 mg/mL
8	bBSA	0.2 mg/ml

spotting pattern:

slide-#: 500

#	spot	Concentration
1-3	MM + DNA
4-6	MM - DNA
7-8	MM + DNA (off-slide cell free expressed, 15 µl reachtion )
10	His-GFP desalt.	~1 mg/mL
11	bBSA	0.2 mg/ml

26.08.2015 (measured)

Flush Protocol:

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	450	1x
BSA	2	60	450	10 mg/ml
Buffer	3	60	450	1x
Anti-GFP (goat, biotinylated)	4	30	600	3 ug/ml
Buffer	5	60	300	1x
StrepCy5	6	30	300	5 ug/ml
Buffer	7	60	300	1x

Only slide 500 was evaluated. Slide 424 and 429 showed very bad surface during measurements which was wiped off → Difficult to evaluate Slide 500: spots 4 and 8 had to be excluded from evaluation.

Slide 500: ROI selection

Slide 500: Binding curves

Measuring cellfree expressed Tetanus antigen

25.08.2015 (spotted) spotting pattern:

slide-#: 216

#	spot	Concentration
1	B+ (1)
2	B+ (2)
3	B+ (3)
4	B- (1)
5	B- (2)
6	K+ (1)
7	His-GFP desalt.	~0.5 mg/mL
8	His-GFP lysate
9	bBSA	0.2 mg/ml
10	K-
11	BSA

slide-#: 208

#	spot	Concentration
1	K+ (1)
2	K+ (2)
3	K+ (3)
4	K- (1)
5	K- (2)
6	B+ (1)
7	His-GFP desalt.	~0.5 mg/mL
8	His-GFP lysate
9	bBSA	0.2 mg/ml
10	B-
11	BSA

26.08.2015 (measured)

Flush protocol:

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (PBS)	1	60	450	1x
BSA	2	60	450	10 mg/ml
Buffer (PBS)	3	60	450	1x
Anti-Tetanus (goat, pk)	4	30	600	25 ug/ml
Buffer (PBS)	5	60	300	1x
Anti-GFP (biotinylated, Goat)	6	30	600	3 ug/ml
Buffer (PBS)	7	60	300	1x
Anti-Goat	8	30	600	5 ug/ml
Buffer (PBS)	9	60	300	1x
StrepCy5	10	30	300	5 ug/ml
Buffer (PBS)	11	60	300	1x

Slide 208: Only 5 spots (spot 3, 5, 7, 8 and 9) could be evaluated.

Slide 208: ROI selection

Slide 216: ROI selection

Slide 208: Binding curves

Slide 216: Binding curves

Measuring own blood: not vaccinated / vaccinated Tetanus samples

21.08.2015 (spotted) spotting pattern:

slide-#: 287, 504

#	spot	Concentration
1-2	Tetanus (11) desalt	460 µg/mL
3-4	Tetanus (11) desalt	100 µg/mL
5-6	Tetanus (11) desalt	20 µg/mL
7-8	His-GFP desalt.	~0.5 mg/mL
9	bBSA	0.2 mg/ml
10-11	Tetanus (11) lysate

22.08.2015 (measured)

Flush protocol slide 287

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (PBS)	1	60	720	1x
BSA	2	60	900	10 mg/ml
Buffer (PBS)	3	60	600	1x
Serum rabbit Anti-GFP	4	30	600	1:330
Buffer (PBS)	5	60	300	1x
Serum (+) SIG002	6	30	600	1:10
Buffer (PBS)	7	60	300	1x
Anti-Human	8	60	300	1,5 ug/ml
Buffer (PBS)	9	60	300	1x

no Strep-Cy5 was flushed over slide –> no scanner Pictures

Flush protocol slide 504

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (PBS)	1	60	720	1x
BSA	2	60	900	10 mg/ml
Buffer (PBS)	3	60	600	1x
Serum (-) SIG002	4	30	600	1:10
Buffer (PBS)	5	60	300	1x
Serum (+) SIG002	6	30	600	1:10
Buffer (PBS)	7	60	300	1x
Anti-Human	8	60	300	1 ug/ml
Buffer (PBS)	9	60	300	1x
Anti-GFP (biotinylated, Goat)	10	60	300	3 ug/ml
Buffer (PBS)	11	60	300	1x
Strep-Cy5	12	60	300	5 ug/ml
Buffer (PBS)	13	60	300	1x

Slide 287: Selection of ROI

Slide 287: Binding curves

Slide 504: Selection of ROI

Slide 504: Binding curves

→ detection of Tetanus antibodys from blood serum after vaccination is possible
→ huge cross reactivity with bBSA spot

Measuring with rabbit serum (immunized with GFP)

18.08.2015 (spotted) 19.08.2015 (measured)

#	spot	Concentration
1-2	His-GFP (cellfree expressed))
3	His-GFP desalt.	0.2 mg/ml
4-5	neg. control (cellfree Expression without DNA)
6-7	His-mCherry lysate	1:10
8-9	His-GFP lysate	1:10
10	BSA	0.2 mg/ml
11	bBSA	0.2 mg/ml

5 Slides Ni-NTA

3X (Slide 208,216,504) Serum(-)/Serum(+),
2x (Slide 500,424) Serum (-)/a-GFP)

2 Slides (315 & 502) PDITC (2x Serum(-)/Serum(+))

Flush protocol Measuring with Serum (-) [not immunized] and Serum (+) [immunized]/a-GFP:

Reagent	#	Flowrate	Priming time	Concentration
Buffer	1	60	780	1x
BSA	2	60	780	10 mg/ml
Buffer	3	60	600	1x
Serum (-)	4	30	600
Buffer	5	60	300	1x
Serum (+)/a-GFP	6	30	600	3 ug/ml
Buffer	7	60	600	1x
anti-Rabbit	8	30	600	5 ug/ml
Buffer	9	60	600	1x
Strep Cy5	10	30	6000	5 ug/ml
Buffer	11	60	600	1x

* 2 ul volume of serum (-) and serum (+) was used for the experiments on PDITC * 5 ul volume of serum (-) and serum (+) was used for the experiments on Ni-NTA

His-GFP lysate quite weak intensity –> maybe too much diluted Kokos cell free expressed GFP works fine on Ni-NTA; does not work on PDITC –> thats good → specific surface

Evaluation (not all slides are shown here):

Ni-NTA Serum (-) / Serum (+)

Slide 208: Selection of ROIs

Slide 208: Binding curves

Slide 502: Selection of ROIs

Slide 502: Binding curves

PDITC Serum (-) / Serum (+)

Slide 315: Selection of ROIs

Slide 315: Binding curves

Slide 502: Selection of ROIs

Slide 502: Binding curves

Ni-NTA (control) Serum (-) / a-GFP

Slide 424: Selection of ROIs

Slide 424: Binding curves

The relative intensity shift for the three approaches (Serum(-)Serum(+) on Ni-NTA ; Serum(-)Serum(+) on PDITC; Serum(-)a-GFP on Ni-NTA) were compared. As expected and seen in the binding curves cellfree expression Mix (His-GFP) shows no signal on PDITC (other proteins within the mix bind to the surface) but on specific Ni-NTA surface. Taken amount of serum performs less (2-3 fold) than 3 ug/ml a-GFP.

Evaluation of the relative intensity shift. From left to right spot 1-11. For each spot rel. intensity shift is plotted with bars for Serum(-)flush and Serum(+)/a-GFP

Emission within 532nm channel (mCherry) after iRIf measurement. Averaged over all samples.

Emission within 635nm channel (Cy5) after iRIf measurement. Averaged over all samples

Measuring cellfree expressed GFP

Spotting (Slide 303):

Spot	Protein	Number of spots	Concentration
1-2	BK foil 1	2	unknown
3-4	neg foil 1	2	unknown
5-6	BK 1	2	unknown
7	GFP-His (Max)	1	0.5 mg/ml
8	GFP-His desalt	1	0.5 mg/ml
9	bBSA	1	0.2 mg/ml
10-11	neg 1	1	unknown

Spotting (Slide 429):

Spot	Protein	Number of spots	Concentration
1-2	BK foil 2	2	unknown
3-4	neg foil	2 2	unknown
5-6	BK 2	2	unknown
7	GFP-His (Max)	1	0.5 mg/ml
8	GFP-His lysate	1	~0.2 mg/ml
9	bBSA	1	0.2 mg/ml
10-11	neg 2	1	unknown

Slide 303: Selection of ROIs

Slide 303: Binding curves

Slide 429: Selection of ROIs

Slide 429: Binding curves

Measuring Halo Slides (GFP dilution series)

Spotting:

Spot	Protein	Concentration
1-2	Halo GFP	0.5 mg/ml
3-4	Halo GFP	0.1 mg/ml
5-6	Halo GFP	0.02 mg/ml
7	bBSA	0.2 mg/ml
8	Halo mCherry	0.5 mg/ml
9	His-GFP (Max)	0.5 mg/ml
10	His Halo GFP (Piehler, pos. control)	0.25 mg/ml
11	His GFP (Piehler, neg. control)	0.25 mg/ml

Flush protocol:

Reagent	#	Flowrate	Priming time	Concentration
Buffer	1	60	720	1x
BSA	2	60	720	10 mg/ml
Buffer	3	60	600	1x
anti-GFP (goat, biot.)	4	30	600	3 ug/ml
Buffer	5	60	300	1x
StrepCy5	6	30	600	5 ug/ml
Buffer	7	60	600	1x

For Evaluation see labjournal surface chemistry

Measuring Kokos cellfree expressed His-GFP on Ni-NTA

14.08.2015

Spotting:

#	spot	Concentration
1-3	HA-GFP-2x6His (cellfree expressed))
4-6	neg. control (cellfree Expression without DNA)
7-8	His-GFP Lysate
9	n. T. GFP Lysate)
10	bBSA	0.2 mg/ml
11	Max GFP	0.5 mg/ml

Flush protocol:

Reagent	#	Flowrate	Priming time	Concentration
Buffer	1	60	780	1x
BSA	2	60	780	10 mg/ml
Buffer	3	60	600	1x
anti HA	4	40	450	5 ug/ml
Buffer	5	60	300	1x
anti GFP	6	40	450	3 ug/ml
Buffer	7	60	600	1x
Strep Cy5	8	40	450	5 ug/ml
Buffer	9	60	600	1x

Roi selection Exp. 46b: Spot 11 couldnt be selected due to air bubble on it

Binding curves of Exp. 46b for all spots

Binding curves of Exp. 46b only for spots 1-6

Spot 11 couldnt be evaluated due to air on it. Spot 1-3 show a-HA binding (weak but clear) and good a-GFP binding → successful cellfree expression No strepcy5 binding can be detected: Tube to flowchamber pulled off just before that step….

Antigens on Ni-NTA

13.08.2015

spotting pattern:

Dilute antigens in TBS (if necessary)!

Dilute antibodies in TBS –> BSA in TBS

2 replicates on PDITC: (024 & 502)

Spot	Protein	Number of spots	Concentration
1-2	pET_1003 (HCV) desalt	2	~0.33 mg/ml
3-4	pET_1703 (HIV) desalt	2	~0.62 mg/ml
5-6	pIG_1501 (Salmonella) desalt	2	~1,24 mg/ml (1:2) ~0.62mg/ml
7	GFP-His (Max)	1	0.5 mg/ml
8	GFP-His desalt	1	0.5 mg/ml
9	GFP-His lysate	1	~0.2 mg/ml
10	bBSA	1	0.2 mg/ml
11	BSA	1	0.2 mg/ml

2 Replicates on NiNTA (501&500)

Spot	Protein	Number of Spots	Concentration
1-2	pET_1003 (HCV) lysate	2	~0.08 mg/ml
3-4	pET_1003 (HCV) desalt	2	~0.33 mg/ml
5	pET_1703 (HIV) lysate	1	~0.15 mg/ml
6	pET_1703 (HIV) desalt	1	~0.62 mg/ml
7	His-GFP (Max)	1	0.5 mg/ml
8	His-GFP lysate	1	~0.2 mg/ml
9	bBSA	1	0.2 mg/ml
10-11	His-GFP aufgereinigt	2	~0.5 mg/ml

2 Replicates on NiNTA (216 & 467)

Spot	Protein	Number of Spots	Concentration
1-2	pET_1703 (HIV) lysate	2	~0.15 mg/ml
3-4	pET_1703 (HIV) desalt	2	~0.62 mg/ml
5	pET_1003 (HCV) lysate	1	~0.08 mg/ml
6	pET_1003 (HCV) desalt	1	~0.33 mg/ml
7	His-GFP (Max)	1	0.5 mg/ml
8	His-GFP desalt	1	~0.5 mg/ml
9	bBSA	1	0.2 mg/ml
10-11	His-GFP lysate	2	~0.2 mg/ml

Flush protocol (for Ni-NTA slides):

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (TBS!!!)	1	60	720	1x
BSA	2	60	600	10 mg/ml
Buffer (TBS!!!)	3	60	600	1x
Anti-HIV (polyclonal,rabbit)	4	30	600	10 ug/ml
Buffer (TBS!!!)	5	60	600	1x
Anti-HCV (polyclonal,rabbit)	6	30	600	10 ug/ml
Buffer (TBS!!!)	7	60	600	1x
Anti-Rabbit (from mouse)	8	30	600	5 ug/ml
Buffer (TBS!!!)	9	60	600	1x
Anti-HIV (monoclonal,mouse)	10	30	600	10 ug/ml
Buffer (TBS!!!)	11	60	600	1x
Anti-HCV (monoclonal,mouse)	12	30	600	10 ug/ml
Buffer (TBS!!!)	13	60	600	1x
Anti-Mouse	14	30	600	5 ug/ml
Buffer (TBS!!!)	15	60	600	1x
Anti-GFP (biotinylated, goat)	16	30	600	3 ug/ml
Buffer (TBS!!!)	17	60	600	1x
StrepCy5	18	30	600	5 ug/ml
Buffer (TBS!!!)	19	60	600	1x

one Ni-NTA slide 1 anti-mouse was changed against anti-his!!! one Ni-NTA slide could not be measured due to hydrophobicity problems

Flush protocol (for PDITC slide 502):

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (TBS!!!)	1	60	720	1x
BSA	2	60	600	10 mg/ml
Buffer (TBS!!!)	3	60	600	1x
Anti-HIV (polyclonal,rabbit)	4	30	600	10 ug/ml
Buffer (TBS!!!)	5	60	600	1x
Anti-HCV (polyclonal,rabbit)	6	30	600	10 ug/ml
Buffer (TBS!!!)	7	60	300	1x
Anti-Rabbit (from mouse)	8	30	600	5 ug/ml
Buffer (TBS!!!)	9	60	600	1x
Anti-Salmonella desalt (13)	10	30	600	~60 ug/ml
Buffer (TBS!!!)	11	60	600	1x
Anti-GFP (biotinylated, goat)	12	30	600	3 ug/ml
Buffer (TBS!!!)	13	60	600	1x
StrepCy5	14	30	600	5 ug/ml
Buffer (TBS!!!)	15	60	600	1x

No antigen/antibody binding (HIV/HCV) was detected on the Ni-NTA slides. Controls worked fine.

Flush protocol (for PDITC slide 24):

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer (TBS!!!)	1	60	720	1x
BSA	2	60	600	10 mg/ml
Buffer (TBS!!!)	3	60	600	1x
Anti-HIV (polyclonal,rabbit)	4	30	600	10 ug/ml
Buffer (TBS!!!)	5	60	600	1x
Anti-HCV (polyclonal,rabbit)	6	30	600	10 ug/ml
Buffer (TBS!!!)	7	60	300	1x
Anti-Rabbit (from mouse)	8	30	600	5 ug/ml
Buffer (TBS!!!)	9	60	600	1x
Anti-Salmonella desalt (13)	10	30	600	~60 ug/ml
Buffer (TBS!!!)	11	60	600	1x
Anti-Salmonella lysate (13)	12	30	600	~70 ug/ml
Buffer (TBS!!!)	13	60	600	1x
Anti-His	14	30	600	20 ug/ml
Buffer (TBS!!!)	15	60	600	1x
Anti-GFP (biotinylated, goat)	16	30	600	3 ug/ml
Buffer (TBS!!!)	17	60	600	1x
StrepCy5	18	30	600	5 ug/ml
Buffer (TBS!!!)	19	60	600	1x

Slide 24: Selection of ROIs: 1 HIV spot (spot 2) and one HCV spot (spot 3) were excluded due to high amounts of salt on it

Slide 24: Binding curves

Slide 502: Selection of ROIs

Slide 502: Binding curves for all spots

Slide 502: Binding curve (second Salmonella spot excluded)

Results: We have specific antibody/antigen binding for Salmonella! (a-HIV/HCV do not bind…) Slide 502: During measurement within the first buffer step/blocking step most of the second Salmonella spot was washed away. Related to this the spot behaves strange. Slide 24: Also anti-salmonella lysate was tested. Here we see (as expected) stronger unspecific binding at other spots (nevertheless greatest binding for the salmonella spots). The unspecific/transient binding proteins get mostly washed away during the following buffer step.

Slide 24: Quotient picture (before a-Salmonella / after a-Salmonella + a-GFP)

Slide 502: Quotient picture (before a-Salmonella / after a-Salmonella + a-GFP)

Cellfree expressed YFP; different GFPs on Ni-NTA

11.08.2015

spotting pattern:

#	spot	concentration
1	His-tYFP-Spy KK
2	His-tYFP-Spy KK + Mg
3	His-tYFP-Spy BK
4	His-tYFP-Spy BK + Mg
5	His-tYFP-Spy neg.
6	bBSA	200 µg/ml
7	Max His-GFP	0.5 mg/mL
8	His-GFP Lysate	~1 mg/mL
9	His-GFP Lysate	~1 mg/mL
10	Max His-GFP	1 mg/mL
11	nT-GFP Lysate	~1 mg/mL

Reagent	#	spot	Elution no.	Concentration
Buffer	1	60	780	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	420	1x
anti-tYFP (rabbit)	4	40	450	20 ug/ml
Buffer	5	60	300	1x
anti-GFP (goat, biotinylated)	6	40	450	3 ug/ml
Buffer	7	60	600	1x
anti-Rabbit	8	40	450	5 ug/ml
Buffer	9	60	300	1x
StrepCy5	10	40	450	5 ug/ml
Buffer	11	60	300	1x

Slide 254: Roi selection

Slide 254: Binding curves. Binding curve shows (better to see with zoom) little a-tYFP (from rabbit) binding to Kokos tYFP spot (Mg2+ fed). Signal is amplified with a-Rabbit

Slide 254: Quotient picture (before/after a-tYFP step)

Slide 254: Quotient picture (before/after a-Rabbit step)

PhyA-Testing auf PDITC

11.08.2015

Spotting:

#	spot	Elution no.	Concentration
1-2	PhyA-GFP
3-4	His-PhyA-GST selbst aufgereinigt	2	6.6 mg/ml → 1:10
5	His-PhyA-GST in AntigenSolution (17)	2	1:1 (1:10 His-PhyA-GST in 5mg/ml BSA : AntigenSolution (1:5)
6	GFP (Max)		1 mg/ml
7	GFP-Lysate-NT
8	His-GFP-Halo (Konstrukt von AG-Roth) - selbst aufgereinigt	?
9	bBSA		0.2 mg/ml
10	GFP (Max) in AntigenSolution (17)	diluted to 1 mg/ml GFP	1:1 (2.0 mg/ml GFP in 5mg/ml BSA (165 ul) : AntigenSolution (1:5) (165 ul))
11	GFP (Max) in Elutionspuffer	diluted to 1 mg/ml GFP	1:1 (2.0 mg/ml GFP in 5mg/ml BSA (165ul) : Elutionsbuffer) (165 ul)

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	780	1x
BSA	2	60	600	10 mg/ml
Buffer	3	60	420	1x
anti-phyA (N-term)	4	40	450	5 ug/ml
Buffer	5	60	300	1x
anti-phyA (rabbit, polyclonal)	6	40	450	5 ug/ml
Buffer	7	60	300	1x
anti-GFP (goat, biotinylated)	8	40	450	3 ug/ml
Buffer	9	60	300	1x
anti-GST	10	40	450	5 ug/ml
Buffer	11	60	600	1x
anti-Rabbit (mouse)	12	40	450	5 ug/ml
Buffer	13	60	300	1x
anti-goat	14	40	450	5 ug/ml
Buffer	15	60	300	1x
StrepCy5	16	40	450	5 ug/ml
Buffer	17	60	300	1x

Duplicates were prepared (Slide 12 and slide 121) but only 1 could be measured, because slide 121 broke ( 8[ ) → Only slide 12 was evaluated.

Slide 12: Selection of ROIs

Slide 12: Binding curves of all spots

Slide 12: Binding curves of PhyA spots

Slide 12: Binding curves of Max GFP spots

Antigen/Antibody

08.08.2015 Spotting:

All spots diluted to proteinconcentration of ~0.4-0.5 mg/ml. Antigens were spinned before spotted.

#	spot	Elution no.	Concentration
1-2	HIV(17)	2	0.4-0.5 mg/ml
3-4	HCV(10)	2	0.4-0.5 mg/ml
5-6	Tetanus(11)	2	0.4-0.5 mg/ml
7	GFP
8	PhyA		0.4-0.5 mg/ml
9	bBSA		0.1 mg/ml
10-11	Salmonella(15)	2	0.4-0.5 mg/ml

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-HIV (polyclonal,rabbit)	4	45	600	10 ug/ml
Buffer	5	60	600	1x
Anti-HCV (polyclonal,rabbit)	6	45	600	10 ug/ml
Buffer	7	60	300	1x
Anti-phyA (N-term) (rabbit)	8	45	600	10 ug/ml
Buffer	9	60	600	1x
Anti-Rabbit	10	30	600	5 ug/ml
Buffer	11	60	600	1x
Anti-HIV (monoclonal,mouse)	12	45	600	10 ug/ml
Buffer	13	60	600	1x
Anti-HCV (monoclonal,mouse)	14	45	600	10 ug/ml
Buffer	15	60	600	1x
Anti-Tetanus (monoclonal,mouse)	16	45	600	10 ug/ml
Buffer	17	60	600	1x
Anti-GFP (biotinylated, goat)	18	30	600	3 ug/ml
Buffer	19	60	600	1x
StrepCy5	20	30	600	5 ug/ml
Buffer	21	60	600	1x
Anti-Mouse	22	30	600	5 ug/ml
Buffer	23	60	600	1x

Again no antibody-binding was detected → We confirmed that in this manner the experiment does not work Binding curves were evaluated, but revealed no further information. Due to the large protocol and some (air)problems (see binding curves of slide 465) during measurement in both cases, curves do not look neat.

Slide 265: Binding curves; some spots were expluded from the evaluation due to air that came on top of them during measurement

Slide 465: Binding curves; this is what it looks like if you do not exclude the spots were air arrived during measurement (spot 2&8) from the evaluation

Testing Halo Slides

06.08.2015

spotting pattern:

#	spot	concentration
1-2	Halo-GFP	1:20
3-4	Halo-GFP	1:10
5-6	Halo-mCHERRY	1:20
7	His-Halo-GFP Piehler
8	His-GFP Piehler
9	bBSA	100 µg/ml
10-11	Halo-mCHERRY	1:10

Slide 426 - measured in old setup

slide wasn't blocked before measurement

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
a-GFP	4	30	600	3 ug/ml
Buffer	5	30	600	1x
StrepCy5	6	30	600	5 ug/ml
Buffer	7	60	600	1x

Slide 424 - measured in old setup (Strep-Cy5 and a-GFP step were accidently switched)

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
a-His	4	30	600	5 ug/ml
Buffer	5	30	600	1x
StrepCy	8	30	600	5 ug/ml
Buffer	9	60	600	1x
a-GFP	6	30	600	3 ug/ml
Buffer	7	30	600	1x

Slide 423 - measured in new setup

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
a-His (from AG Roth)	4	30	600	5 ug/ml
Buffer	5	30	600	1x
a-GFP	6	30	600	3 ug/ml
Buffer	7	30	600	1x
StrepCy	8	30	600	5 ug/ml
Buffer	9	60	600	1x

Slide 466 - measured in old setup

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
a-His (from iGEM Lab)	4	30	600	5 ug/ml
Buffer	5	30	600	1x
a-GFP	6	30	600	3 ug/ml
Buffer	7	30	600	1x
StrepCy	8	30	600	5 ug/ml
Buffer	9	60	600	1x

For evaluation of Halo-Experiment → look up in labjornal of the surface chemistry

Spotting antibodies to flush with purified antigen lysate

04.08.2015

spotting pattern:

#	spot (AB)	AB-Concentration [µg/mL]
1-2	HIV: gp41 DDX1306 (mouse)	100
3-4	HCV: HCV-AB (mouse)	100
5-6	Tetanus: HYB 278-01 (mouse)	100
7-8	His-GFP	1000
9	bBSA	100 mg/mL
10-11	a-Salmonella-(pIG15_1301)	100

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	60	940	1x
BSA	2	30	900	10 mg/ml
Buffer	3	30	600	1x
HIV (17)	4	20	900	0.25 mg/ml
Buffer	5	30	600	1x
HCV (10)	6	20	900	0.25 mg/ml
Buffer	7	30	600	1x
Tetanus (11)	8	20	900	0.25 mg/ml
Buffer	9	30	600	1x
Anti-Mouse	10	20	900	5 ug/ml
Buffer	11	30	600	1x
Anti-GFP	12	20	900	3 ug/ml
Buffer	13	30	600	1x

Only 1 slide was measured. Second slide syringe didnt suck solutions. Results: Antigens didnt bind to the immobilized antibodies. Strangely, anti-mouse only bound to a-Tetanus spot, and not to a-HIV and a-HCV spots, even though they are from mice, too.

Slide 29: Selection of ROIs

Slide 29: Binding curve

Measuring HIV,HCV,Tet,Sal with polyclonal and monoclonal ABs

31.07.2015

In contrast to the previous experiments 15 (Salmonella) and 11 (Tetanus) were heatshocked just before spotting (to denature the antigens → maybe AB binds better to the denatured antigens (the primary structure)!?)

#	spot	Elution no.	Concentration
1-2	HIV(17)	1	1.0 mg/ml
3-4	HCV(10)	1	0.74 mg/ml
5-6	Tetanus(11)	1	3.75 mg/ml
7-8	GFP		1.0 mg/ml
9	bBSA		0.1 mg/ml
10-11	Salmonella(15)	1	6.7 mg/ml

Flush protocol (Slide 208):

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	80	940	1x
BSA	2	45	600	10 mg/ml
Buffer	3	60	600	1x
Anti-GFP(goat;biotinylated)	4	30	600	5 ug/ml
Buffer	5	60	300	1x
Anti-HIV (monoclonal)	6	20	900	20 ug/ml
Buffer	7	60	300	1x
Anti-HCV (monoclonal)	8	20	900	20 ug/ml
Buffer	9	60	300	1x
Anti-Tetanus (monoclonal)	10	30	600	20 ug/ml
Buffer	11	60	300	1x
Anti-HIV (polyclonall)	12	20	900	20 ug/ml
Buffer	13	60	300	1x
Anti-HCV (polyclonal)	14	20	900	20 ug/ml
Buffer	15	60	300	1x
Anti-Salmonella (1301 Elu1)	16	20	900	1:3
Buffer	17	60	300	1x
….further steps were skipped

For second slide flush protocol was changed→ added anti-His step and further dilutions of anti-Salmonella (but threw out a-HIV/a-HCV/anti-Tet) Flush protocol (Slide 12):

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(goat;biotinylated)	4	30	600	5 ug/ml
Buffer	5	60	300	1x
Anti-His	6	30	600	20 ug/ml
Buffer	7	60	300	1x
Anti-Salm (Elu 3)	8	20	900	1:5
Buffer	9	60	300	1x
Anti-Salm (Elu 3)	10	30	600	1:2
Buffer	11	60	300	1x
Anti-Salm (Elu 1)	12	30	600	1:10
Buffer	13	60	300	1x
Anti-Salm (Elu 1)	14	20	900	1:3
Buffer	15	60	300	1x
StrepCy5	16	30	600	5 ug/ml
Buffer	17	60	300	1x

Results: Anti-His at least confirmed that something with a His-Tag is within the HIV and HCV spots (see binding curves of slide 12).

Slide 208: Binding curves; flushing with anti-Salmonella lysate screwed up the measurement → protocol only to this step

Slide 208: Selection of ROIs: As can be seen we faced some problems during that measurement ;)

Slide 12: Binding curves

Slide 12: Selection of ROIs

03.08.2015 Flush protocol:

Reagent	Step	Flow rate (ul/min)	Priming time	Concentration
Buffer	1	80	940	1x
BSA	2	45	600	10 mg/ml
Buffer	3	60	600	1x
Anti-GFP(goat;biotinylated)	4	25	720	5 ug/ml
Buffer	5	60	300	1x
Anti-tYFP (rabbit)	6	15	1200	20 ug/ml
Buffer	7	60	300	1x
Anti-PhyA (rabbit)	8	20	900	20 ug/ml
Buffer	9	60	300	1x
Anti-Rabbit	10	30	600	5 ug/ml
Buffer	11	60	300	1x
StrepCy5	12	30	600	5 ug/ml
Buffer	13	60	300	1x

Results: Binding of anti-phyA to phyA was not observed in RIfs. Yet, at both measurements was faced some problems: much of the protein was washed away from the spotted spot. → We have to take lower concentrations for spotting (100-400 ug/ml) Anti-tYFP to YFP binding could not be detected aswell: Actually, this wont work on PDITC since all of the expression mix proteins also bind to the spot, compete with the (already low concentration) YFP for binding → almost no YFP can bind to the spot. We have to repeat experiment for cellfree expressed YFP/GFP with specific surface (e.g. Ni-NTA)

Slide 303: ROI selection

Slide 303: binding curves

Slide 303: Inverted QP during initial buffer step: Shows that already much of the PhyA is flushed away from the spot

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Difference between revisions of "Team:Freiburg/Labjournals/irif/August"

Latest revision as of 08:13, 10 September 2015

iRIf Labjournal August

Distinguish between different antigen/antibody interaction (GFP, mouse, rabbit) (on PDITC) [finished]

Affibody (Stockholm cooporation)

Measuring own blood (Tet (-) / Tet (+)) on PDITC

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

Measuring own blood (Tetanus unimmunized vs. immunized) on PDITC

Measuring on-slide cellfree expressed GFP (Kokos Mix, AG Roth Mix, Reticolozytes Mix)

Measuring on-slide cellfree expressed GFP

Measuring cellfree expressed Tetanus antigen

Measuring own blood: not vaccinated / vaccinated Tetanus samples

Measuring with rabbit serum (immunized with GFP)

Measuring cellfree expressed GFP

Measuring Halo Slides (GFP dilution series)

Measuring Kokos cellfree expressed His-GFP on Ni-NTA

Antigens on Ni-NTA

Cellfree expressed YFP; different GFPs on Ni-NTA

PhyA-Testing auf PDITC

Antigen/Antibody

Testing Halo Slides

Spotting antibodies to flush with purified antigen lysate

Measuring HIV,HCV,Tet,Sal with polyclonal and monoclonal ABs