Aug 27, 2024
WesternBlot Paramecium bursaria (Pb)
- 1University of Oxford
Protocol Citation: Estelle Kilias 2024. WesternBlot Paramecium bursaria (Pb). protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1q58kgr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 26, 2023
Last Modified: August 27, 2024
Protocol Integer ID: 89921
Abstract
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.
Protein extraction
Protein extraction
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
10m
Centrifuge the strained culture for 10 min at 800xg
10m
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
3m
Centrifuge the washed culture for 10 min at 800xg
10m
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.
3m
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)
10m
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
3m
Incubate the sample at 37C for 30min
30m
Optional: Make dilutions of your sample as needed using pre-warmed 6xSDS loading buffer (37C)
Optional: Centrifuge at max speed for 5 min and use supernatant to load the gel
Protein separation
Protein separation
Prepare 1x Running buffer (BioRad; Cat. #1610732)
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.
Load the gel using 7ul for the protein ladder and 12 ul of protein extract. Fill empty wells with loading buffer
Run the gel for 60 min at 100V
1h
Transfer
Transfer
Prepare 1x Transfer buffer, add 20% methanol (BioRad; Cat.# 1610734) and store at 4C.
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
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
15m
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.
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.
Run for 60 min at 100V
1h
Staining and blocking
Staining and blocking
Carefully remove the transfer membrane and rinse it in a square petri dish with ultrapure water.
Incubate the membrane with Pierce Reversible Protein Stain (ThermoScientific, Cat.# 24580) for 4 min
4m
Remove the stain and rinse the membrane 1x quick with Pierce Destain solution
Remove the Pierce Destain solution and rinse the membrane 2x with ultrapure water
Incubate the membrane for 5 min in Pierce Destain solution
5m
Remove the Pierce Destain solution and rinse the membrane 4x with ultrapure water.
Take a picture of the membrane.
Incubate the membrane for 5 min in ultrapure water
5m
Incubate the membrane for 2 min in Pierce Stain Eraser or until membrane is unstained
2m
Remove the Pierce Stain eraser and rinse the membrane 4x with ultrapure water
Incubate the membrane for 5 min in ultrapure water
5m
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).
15m
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
1m
Incubate in 50 ml EveryBlot Blocking buffer (BioRad, Cat.# 12010020) overnight at 4C on rotor.
Antibody incubations
Antibody incubations
Wash membrane with PBS-Tween (1xPBS + 0.1% Tween-20)
PBS: Sigma Aldrich P4417-50Tab
Tween-20:Sigma Aldrich P1379-250ml
15m
Incubate membrane in 5 ml EveryBlot Blocking buffer (BioRad, Cat.# 12010020) with antibody#1 [1:250] for 3 hrs on the rotor
3h
Wash membrane with PBS-Tween (1xPBS + 0.1% Tween-20)
PBS: Sigma Aldrich P4417-50Tab
Tween-20:Sigma Aldrich P1379-250ml
15m
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
1h
Washing and staining
Washing and staining
30m
30m
Wash membrane in 5 ml PBS-Tween (1xPBS + 0.01% Tween-20), shake for 15 min at room temperature
15m
Wash in 2 ml PBS-Tween (1xPBS + 0.01% Tween-20) and shake for 5 min at room remperature
5m
Wash in 2 ml PBS and shake for 5 min at room temperature
5m
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)
5m
Image membrane