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Appendix
Expression & affinity analysis of recombinant RX against pathogenic α-synuclein
Additional material supplementary to the main report is shown in this document. Not all material produced during the study time is included, only the most significant material helpful to understand the study considered not necessary to include in the main report. Concentrations and plate layout for ELISAs are included in the Appendix.
Page Blue protein staining protocol
1- Place the gel in an uncovered microwaveable tray containing 100mL of ultrapure water and microwave for 60 seconds
2- Remove tray from the microwave, gently agitate for 4 minutes and discard water
3- Repeat the above steps two additional times.
4- Discard the last wash and add 20mL of PageBlue Protein Staining Solution, or sufficient volume to cover the gel, and microwave on high power for 20-30 seconds (Do not allow the reagent to boil).
5- Gently agitate the gel in the staining solution for an additional 20 minutes.
6- Discard staining solution and rinse the gel two times with ultrapure water.
7- Wash the gel for 5 minutes with 100-200mL of ultrapure water.
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Figure A1. Coomassie gel for concentrated D123mut F6. D123mut F2 is used as a reference for the purity.
Expected size of D123mut is 40.4 kDa and the bands at ~55 kDa are believed to represent D123mut.Figure A2. Trimerization ratio optimization. Doubled and halved ratios of SpyTag to SpyCatcher were tested on Native page gels. The left image is with only Strep-Tag detection and the right image is with Strep-Tag detection
and His-tag detection. Expected size for D123mut F2 is 40.4 kDa, for TriCatcher-GFP is 60.5 kDa, for SpyCatcher-o-tri 61.8, for Trimerized GFP 181.7 kDa and for Trimerized o-tri 183.0 kDa.
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Table A1. Layout and concentrations of the 96 half area plate used in ELISA-1. 64 wells of the plate used. All fractions are from D123mut. Coating was for all indirect ELISAs 500 ng/ml.
1.
F1
2.
F1
3.
F2
4.
F2
5.
F6
6.
F6 7.
Syn-O2 8.
Syn-O2
A 1000 nM 1 nM
B 200 nM 0.2 nM
C 40 nM 0.04 nM
D 8 nM 0.008 nM
E 1.6 nM 0.0016 nM
F 0.32 nM 0,00032 nM
G 0.064 nM 0.000064 nM
H Blank Blank
Figure A3. Showing results of concentration optimization ELISA with SynO2 as positive control on a logarithmic x-axis and non-logarithmic y-axis. D123mut F6 wash at different concentrations was the only
fraction used. KD for Syn-O2 was 0.003 -0,5
0 0,5 1 1,5 2 2,5 3
0,00001 0,001 0,1 10 1000
Absorbance
RX concentration nM
RX concentration optimization
D123mut F6 D123mut F6 D123mut F6 Syn-O2
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Figure A4. Showing results from anti strep binding indirect ELISA. The x-axis displays the concentration of anti-strep antibody in a 1:X dilution series. Ref is the reference protein used that was known to bind a-strep well.
Figure A5. D123 OD and concentrations in a Scatchard plot. The concentration used in the Y-axis is the total concentration of D123 that was added to the wells of the ELISA plate. The data points show
non-linearity disabling the plots use to determine K
Dfor D123.
0 0,5 1 1,5 2 2,5 3
1 10 100 1000 10000 100000
Absorbance
Dilution relationship
anti-strep ab dilution optimization
a-strep - D123mut a-strep - Ref
R² = 0,1214
-0,5 0 0,5 1 1,5 2
0 0,5 1 1,5 2 2,5
OD/Conc (nM)
OD
Scatchard plot
D123 Linear (D123)
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Figure A6. Competitive ELISA of RX binding to α-syn monomers and HNE-induced oligomers. HNE-induced oligomers were coated in a serial 3:1 dilution starting at 70 000 nM. Incubated monomers and oligomers had a
constant concentration of 980 µg/ml. RX had a constant concentration of 500 nM.
Table A2. Volumes of added samples for Coomassie staining and western blot for the September transfections.
D1 Western 0.5 µg
F1
Sample: 2.63 PBS: 9.37
Sample buffer: 4.00 Red agent: 1.60 Tot: 16.00
F2+3
Sample: 13.24 PBS: 0.00
Sample buffer: 4.41 Red agent: 1.76 Tot: 17.60 D1
Coomassie 1 µg
F1
Sample: 5.26 PBS: 6.74
Sample buffer: 4.00 Red agent: 1.60
F2+3
Sample: 26.48 PBS: 0.00
Sample buffer: 8.83 Red agent: 3.52
-0,02 0 0,02 0,04 0,06 0,08 0,1
1 10 100 1000 10000 100000
Absorbance
Coated oligomer conc nM
a-syn competitive ELISA
Monomers Oligomers
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Tot: 16.00 Tot: 35.20
D123mut Western 0.5 µg
F1
Sample: 0.84 PBS: 7.78
Sample buffer: 4.00 Red agent: 1.60 Tot: 16.00
F2
Sample: 1.29 PBS: 10.71
Sample buffer: 4.00 Red agent: 1.60 Tot: 16.00
F3+4+5
Sample: 1.43 PBS: 10.57
Sample buffer: 4.00 Red agent: 1.60 Tot: 16.00 D123mut
Coomassie 1 µg
F1
Sample: 1.69 PBS: 10.31
Sample buffer: 4.00 Red agent: 1.60
F2
Sample: 2.59 PBS: 9.41
Sample buffer: 4.00 Red agent: 1.60
F3+4+5
