Purification of α-synuclein

Materials required

Sl. No. Chemicals/Reagents Availability

1 pRK172/PET28a+ vector with alpha-syn constructs +
2 BL21 DE3 competent cells +
3 100/500/1000 mL LB broth +
4 80% Glycerol +
5 1M IPTG (1000x stock) +
6 100mg/mL Ampicillin 50mg/mL Kanamycin stock +
7 50mM Tris pH8 +
8 10mM EDTA +
9 150mM NaCl +
10 10% Streptomycin sulphate +
11 Glacial acetic acid +
12 Saturated ammonium sulphate (4.1M) +
13 100mM ammonium acetate +
14 Ethanol +
15 10mM HEPES pH7.4 +
16 145mM KCl +
17 0.04% Sodium azide +
18 1M KOH +
19 Autoclaved milliQ water +

Sl. No. Other Materials/Instruments Availability

1 37⁰C incubator +
2 UV spectrophotometer +
3 Cryo tubes +
4 -80⁰C freezer +
5 Cold centrifuge +
6 Falcon tubes +
7 Water bath +
8 Eppendorf tubes +
9 Ice +
10 pH meter/strips +
11 PD10-G10 column +
12 AMICON centrifugal filter 10KDa +
13 Scalpel +
14 Spirit lamp +

Solution/Buffer preparation

 TNE buffer

Prepare TNE buffer containing 50mM Tris pH8 (6.057g/L), 10mM EDTA (3.7224g/L) and 150mM
NaCl (8.766g/L).

Adjust the pH to 8 using concentrated HCl.

Filter using a syringe/vacuum filter.

  HBS buffer

Prepare HBS buffer containing 10mM HEPES (2.383g/L), 145mM KCl (10.8097g/L) and 0.04%
sodium azide (0.4g/L).

Adjust the pH to 7.4 using 1M KOH.

Filter using a syringe/vacuum filter.

 Saturated Ammonium sulphate solution (prepare fresh)

The concentration of saturated ammonium sulphate solution at 4⁰C is 3.93M.

Prepare the required volume at 4⁰C only, keeping it on ice or in cold room.

 Thioflavin T stock

To prepare ThT stock, dissolve around 1-2 mg of it in 50mM Glycine-NaOH buffer (pH 8.5).

Check for the purity of ThT in the product datasheet.

Multiply the extension coefficient of ThT (36000 m-1cm-1) by the percentage of purity given in the
datasheet. Use this value for calculating the concentration of ThT.

Measure the absorbance value of ThT(1μl) in 50mM Glycine-NaOH buffer (500μl) at 412nm and
calculate the concentration using Beer Lambert’s law equation.

Procedure

1. Transform pRK172 ba
2. cterial expression vector containing α-synuclein (wild type) into BL21 competent cells.
3. Pick a single colony and expel it into 5mL autoclaved LB Broth with Ampicillin (5uL of
100mg/mL stock). Incubate it at 37⁰C for overnight.
4. Transfer 5mL of this starter culture to 500mL LB Broth in a conical flask (1% of secondary
culture volume).
5. Incubate it at 37⁰C until the OD value reaches 0.7-1.5. (collect 10uL-1000uL and prepare un-
induced total cell extract)
(At this step glycerol stocks can be prepared and stored at -80⁰C)
6. Add 1mM IPTG (100uL of 1000x stock) to the 500mL LB Broth culture and incubate at 37⁰C
for 3 hours. (collect 10uL-1000uL and prepare induced total cell extract)
7. Transfer the culture to two 50mL falcon tubes and pellet down the cells at 6000 rpm for 10
minutes at 4⁰C.
8. Now re-suspend the cells in 1000uL of TNE buffer per 100 mL culture and transfer it to a
fresh 2 mL eppendorf tubes.
9. Aliquots can be prepared and stored at -80⁰C until purification.
10. Place the tube with cells in thermo-block for 10 minutes immediately after taking it out from
-80⁰C.
11. Pellet down the cells at maximum rpm for 10 minutes at 4⁰C.
12. Transfer only the supernatant to a fresh 2mL eppendorf tube.
 (Keep aside 10uL of supernatant for SDS PAGE)
13. Add 136uL of 10% streptomycin sulphate and 288uL of glacial acetic acid per 1 mL of
supernatant.
14. Gently mix and keep it on ice for 30 minutes.
15. Spin the mixture at the maximum rpm for 10 minutes at 4⁰C.
16. Transfer the supernatant to a fresh tube.
(Keep aside 10-20uL of supernatant as well as the pellet for SDS PAGE)
17. Precipitate the protein by adding equal volume of saturated ammonium sulphate at 4⁰C for 30
minutes.
18. Spin at maximum rpm for 10-20 minutes at 4⁰C.
19. Remove the supernatant.
(Don’t discard. Can be checked using SDS PAGE)
20. Wash the pellet with 1mL diluted ammonium sulphate solution (1:1 ratio of saturated
ammonium sulphate and water) at 4⁰C.
21. Re-suspend the pellet in 450uL of 100mM ammonium acetate (forms a cloudy solution).
22. Add equal volume of ethanol to precipitate the protein at room temperature.
23. Centrifuge at 10,000 rpm for 5 minutes.
24. Discard the supernatant and dry the pellet to make sure it is completely free from any residual
ethanol.
(Steps 20-22 can be repeated twice)
25. Re-suspend the pellet in suitable volume of 100mM ammonium acetate.
26. Now take a PD10 column and cut the bottom end of it with a heated scalpel. (column volume
is 15mL)
27. Wash the column with milliQ water.
28. Then wash it with the buffer (4 times the volume of the column) in which the protein is to be
eluted (in this case HBS buffer is used).
29. Add the protein sample to the column and start collecting the droplets from the column (10
drops in 1 tube). Once the protein sample enters the column completely add HBS buffer again
to the column.
30. Run the collected fractions in an SDS PAGE gel to identify the fractions containing purified
protein. The protein samples can be stored at -80⁰C for future use.
31. Do SDS PAGE to check the homogeneity of the purified protein.

Concentration of protein
1. Collect the fractions containing pure α-synuclein protein in a centrifugal filter tube which has
a column with pores of size 10KDa.
2. Centrifuge the tube at 3000 rpm 4⁰C, until the protein sample is concentrated in a desired
volume of buffer.
3. Estimate the protein concentration using a suitable method (we use Urea method)

Protein estimation using Urea method

1. Prepare 10M Urea stock in autoclaved distilled water (has to be prepared fresh every time).
2. For a 200μL reaction, take 1/5th of the volume of protein sample (depends on how
concentrated the protein is) and 8M final concentration urea.
3. Incubate at room temperature for 40 minutes.
4. Exactly at end of 40 minutes incubation start measuring the absorbance value at 280nm.
5. Calculate the concentration using Beer Lambert’s law equation.

Pre-formed Fibril synthesis

1. Filter the protein sample through 0.22μm syringe filter to remove any particulate matter or
bigger aggregates.
2. Add 0.15mM SDS (final concentration) to 1mL of protein sample containing 1mg of protein
in a 10ml flat base glass tube.
3. Incubate it at 37⁰C for 12-16 hours with constant stirring at 400 rpm (using magnetic stirrer of
~1cm).
4. Collect 10μl of this sample at different time points and proceed with analysis immediately or
store at -80⁰C.
5. Analyse the fibril formation using Thioflavin T assay.

Thioflavin T assay
1. Prepare 10μM ThT in 50mM Glycine-NaOH buffer (pH 8.5) from ThT stock.
2. Add 10μl of each of the collected samples to 1mL of 10μM ThT in 50mM Glycine-NaOH
buffer.
3. Measure the fluorescence intensity at 485 nm upon exciting the samples at 445 nm.