1.Plates: Nunc-Immuno 96-Well Plates (Maxisorp Surface Treatment) (Fischer Cat. No. 12-565-136)
Primary: Monoclonal mAb211, 1:1000 dilution for coating, store at 2-8°C
Secondary: Biotinylated mAB211, 1:1000 dilution for incubation, store at 2-8°C
3. 30% Hydrogen Peroxide (H2O2) (Fischer Cat. No. H325-100; $62.44 for 100ml bottle)
4. o-Phenylenediamine dihydrochloride (OPD) (1mg tablets) (Sigma Product No. P6662-50TAB; $150.50 for 50 tablets)
5. 96-well microtiter plate (Fischer Cat. No. 12-565-136; $183.25 for 60 plates)
6. 1N Sulfuric acid (Fischer Cat. No. 83004; $8.89 for 120ml bottle)
B.Buffers and Solutions
1.Coating Buffer: Carbonate/bicarbonate (pH 9.6) 100ml
**Use carbonate/bicarbonate to adjust pH to 9.6**
2.PBS-Tween (PBST): 0.05% (v/v) Tween-20 in PBS (50μl Tween-20 in 100ml PBS)
3.PBST-BSA: PBS w/ 0.05% (v/v) Tween-20 and 0.5% (w/v) BSA (250mg BSA in 50ml PBST)
4.Phosphate-Citrate: 50mM citric acid and 100mM sodium phosphate
0.74g Dibasic sodium phosphate and 1.3g Citric acid
Dissolve in 50ml water
Adjust pH to 5.0
Add 40ul fresh 30% H2O2 to 100ml solution before use
5.Blocking: 0.5% (w/v) BSA in PBS 500mg BSA 100ml PBS
6.HRP substrate: (OPD) in phosphate-citrate buffer
Dissolve 1mg OPD tablet in 2.5ml buffer
Vortex to mix
Do not touch tablet with fingers or metallic forceps
7.Stopper: 1N Sulfuric Acid
8-well multichannel pipette
Airtight sandwich box
1 Dilute mAB211 1:1000 in Coating Buffer to approximately 2.5 μg/ml.
2 Add 100ul of mAB211 dilution from step 1 to each well of microtiter plate. Incubate overnight (o/n) @ 4°C sealed plate sealer.
3 Wash plates 3x w/ PBST. Aspirate and blot dry with Kimwipe.
4 Store individually in plastic bags @ -20°C. Plates may be stored for 15d.
E. Preparation of α-synuclein standards
1.Get one 11uL aliquot from preformed α-synuclein fibrils stock
2.Concentration of α-synuclein in fibrils = 1ug/uL
3.Dilute the stock 30X
4.Add 319uL PBS (total volume = 330uL)
5.Sonicate stock for 10 minutes at 1 minute intervals
6.Sonication is done in intervals to prevent heating of solution. The energy provided by the heat can induce the sonicated fragments to reform into fibrils
7.Make 5 serial dilutions
8.Add 30uL of stock into 300uL PBS and so on
1. Thaw plates before opening bags to prevent condensation in the wells.
2. Add 200ul Blocking Buffer to each well and incubate 1 hour @ room temperature (RT).
3. Wash 3x w/ PBST. Aspirate and blot dry.
4. Prepare standards and samples in PBS.
5. Add 100ul of dilutions to wells.
6. Incubate plate 2-3h @ RT.
7. Incubate o/n @ 4C.
8. Wash 3x w/ PBST. Aspirate and blot dry.
9. Dilute biotinylated mAB211 in PBST + 0.5% Bovine Serum Albumin (BSA).
10. Add 100ul diluted Ab to each well. Incubate 2h @ RT.
11. Wash 3x w/ PBST. Aspirate and blot dry.
12. Prepare HRP-Streptavidin......
13. Add 100ul diluted HRP-Streptavidin to each well. Incubate 2h @ RT.
14. Wash 3x w/ PBST. Aspirate and blot dry.
15. Prepare Peroxidase Substrate immediately before use.
16. Add 100ul Peroxidase Substrate to each well and incubate 10min in the dark @ RT.
17. Add 100ul Stopper Solution to wells and read plate at 490nm.