TITLE: WesternBlot Paramecium bursaria (Pb)
AUTHORS: Estelle Kilias

[DESCRIPTION]
Western Blot protocol for Pb including all steps from the cell harvest to the final ECL stain. Culture density is an important factor that requires checking prior starting.

[STEPS]
SECTION: Protein extraction
1. To concentrate the culture, strain 100 ml of a dense Paramecium culture through a 40 micrometer strainer and split the volume over two 50 ml falcon tubes

SECTION: Protein extraction
2. Centrifuge the strained culture for 10 min at 800xg 
SECTION: Protein extraction
5. Remove the supernatant from each falcon tube but leave 1 ml to avoid disturbing the pellet. Transfer the 1 ml into 1.5ml Eppendorf tubes.
SECTION: Protein extraction
6. Centrifuge the sample for 10 min at 800 xg  and remove the supernatant. Leave 50 ul to not disturb the pellet and pool the 2 tubes. You have now concentrated the sample 1:1000 (endvolume: 100 ul) 
SECTION: Protein extraction
7. Add 200 ul of pre-warmed 2x SDS buffer (37C) supplemented with Proteinase Inhibitor (Roche; Cat. 4693116001), 2ul RNAseA (Qiagen; Cat. 19101) and 2ul DNAseI (Fisher Scientific; Cat. EN0521) to your concentrated sample
SECTION: Protein extraction
8. Incubate the sample at 37C for 30min

SECTION: Protein extraction
9. Optional: Make dilutions of your sample as needed using pre-warmed 6xSDS loading buffer (37C)
SECTION: Protein extraction
10. Optional: Centrifuge at max speed for 5 min and use supernatant to load the gel
SECTION: Protein separation
11. Prepare 1x Running buffer (BioRad; Cat. #1610732)
SECTION: Protein separation
13. Load the gel using 7ul for the protein ladder and 12 ul of protein extract. Fill empty wells with loading buffer
SECTION: Protein separation
14. Run the gel for 60 min at 100V
SECTION: Transfer
15. Prepare 1x Transfer buffer, add 20% methanol (BioRad; Cat.# 1610734) and store at 4C.
SECTION: Transfer
17. Soak the transfer membrane (Nitrocellulose Membrane, 0.2um BioRad Cat. #1620112), sponge, filter paper (Mini Trans-Blot Cat. #1703932) and gel in 1x transfer buffer for 15min on the shaker 30rpm at room temperature
SECTION: Transfer
16. Remove gel from holder and rinse under water. Use spatula to crack open each edge carefully. Optional: Cut a corner to mark the start of your gel
SECTION: Transfer
18. Assemble the 'transfer-cassette' and try to avoid any bubles. Use the small roller between each step carefully. Sequence: sponge/filter paper/gel/transfer membrane/filter paper/sponge. When closing the cassette try not to put to much pressure.
SECTION: Transfer
19. Put gel electrophoresis chamber into a tray on the magnetic stirrer. Fill the chamber with ice cold transfer buffer and add a spinbar to allow the buffer to move during the transfer. Fill the tray with ice to keep it cold during running and load the cassette into the chamber. 
SECTION: Transfer
20. Run for 60 min at 100V
SECTION: Staining and blocking
21. Carefully remove the transfer membrane and rinse it in a square petri dish with ultrapure water. 
SECTION: Staining and blocking
22. Incubate the membrane with Pierce Reversible Protein Stain (ThermoScientific, Cat.# 24580) for 4 min
SECTION: Staining and blocking
23. Remove the stain and rinse the membrane 1x quick with Pierce Destain solution
SECTION: Staining and blocking
24. Remove the Pierce Destain solution and rinse the membrane 2x with ultrapure water
SECTION: Staining and blocking
25. Incubate the membrane for 5 min in Pierce Destain solution 
SECTION: Staining and blocking
26. Remove the Pierce Destain solution and rinse the membrane 4x with ultrapure water. 
Take a picture of the membrane.
SECTION: Staining and blocking
27. Incubate the membrane for 5 min in ultrapure water
SECTION: Staining and blocking
28. Incubate the membrane for 2 min in Pierce Stain Eraser or until membrane is unstained
SECTION: Staining and blocking
29. Remove the Pierce Stain eraser and rinse the membrane 4x with ultrapure water
SECTION: Staining and blocking
30. Incubate the membrane for 5 min in ultrapure water
SECTION: Staining and blocking
31. Incubate the membrane for 15 min in Denaturation Buffer (pre-warmed at 55C) 
Denaturation buffer (50 mL): 10 mL 10% SDS, 350 uL beta-mercaptoethanol, 6.25 mL 0.5 M Tris HCl, 33 mL H2O (incubate buffer in 55C water bath beforehand). 
SECTION: Staining and blocking
32. Incubate the membrane for 1 min in PBS-Tween (1x PBS + 0.1% Tween-20)
PBS: Sigma Aldrich P4417-50Tab
Tween-20:Sigma Aldrich P1379-250ml
SECTION: Staining and blocking
33. Incubate in 50 ml EveryBlot Blocking buffer (BioRad, Cat.# 12010020) overnight at 4C on rotor.
SECTION: Protein separation
12. Fill the gel chamber with '1x Running buffer'. Take a pre-made Mini PROTEAN TGX gel (BioRad Cat.# 4561096), rinse it briefly with water and load it into the holder. Carefully remove the comb. Remember to remove the green tape at the bottom. 
SECTION: Antibody incubations
34. Wash membrane with PBS-Tween (1xPBS + 0.1% Tween-20)
PBS: Sigma Aldrich P4417-50Tab
Tween-20:Sigma Aldrich P1379-250ml
SECTION: Antibody incubations
35. Incubate membrane in 5 ml EveryBlot Blocking buffer (BioRad, Cat.# 12010020) with antibody#1  [1:250] for 3 hrs on the rotor

SECTION: Antibody incubations
36. Wash membrane with PBS-Tween (1xPBS + 0.1% Tween-20)
PBS: Sigma Aldrich P4417-50Tab
Tween-20:Sigma Aldrich P1379-250ml
SECTION: Antibody incubations
37. Incubate membrane in 5 ml EveryBlot Blocking buffer (BioRad, Cat.# 12010020) with antibody#2 [1:5000] for 1 hr in the dark on the rotor at room temperature
SECTION: Washing and staining
38. Wash membrane in 5 ml PBS-Tween (1xPBS + 0.01% Tween-20), shake for 15 min at room temperature
SECTION: Washing and staining
39. Wash in 2 ml PBS-Tween (1xPBS + 0.01% Tween-20) and shake for 5 min at room remperature
SECTION: Washing and staining
40. Wash in 2 ml PBS and shake for 5 min at room temperature
SECTION: Washing and staining
41. Prepare Pierce ECL dye mix (2.5 ml of each solution) and stain membrane for 5 min on shaker.
Pierce ECL Western (ThermoScientific Cat.#: 32106)
SECTION: Protein extraction
3. Remove the supernatant from each falcon tube and wash the pellet with 5 ml of autoclaved NCL media. 
NCL media: https://doi.org/10.1073/pnas.210887411
SECTION: Protein extraction
4. Centrifuge the washed culture for 10 min at 800xg 
SECTION: Washing and staining
42. Image membrane