TITLE: Protein Extraction and Western Blot of Human Lung Organoids 
AUTHORS: Morris Baumgardt, Maren Hülsemann, Diana Fatykhova, Stefan Hippenstiel, Andreas C. Hocke, Katja Hönzke

[DESCRIPTION]
This protocol describes the protein extraction from human alveolar-like organoids followed by western blotting. It gives a detailed description of the preparation of cell lysate from human alveolar-like organoids, followed by exact steps to perform a western blot and the immunostaining to detect ACE2, TMPRSS2, and FURIN as host factors of SARS-CoV-2.

[BEFORE_START]
Please use protein low-bind tubes for all steps.

[GUIDELINES]
This protocol describes the processing of human alveolar-like organoids which have been grown according to Youk et al., 2020. https://doi.org/10.1016/j.stem.2020.10.004.

[STEPS]
SECTION: Prepare cell lysate of alveolar-like organoids
1. You need a sufficient amount of organoids (approx. 300,000 cells per well) and organoids should be collected from minimum two wells of a 24-well plate (total ~600,000 cells).
SECTION: Prepare cell lysate of alveolar-like organoids
2. From here all steps on ice. 

Keep tubes, reagents and buffers on ice.


SECTION: Prepare cell lysate of alveolar-like organoids
3. Remove organoid media by aspiration.
SECTION: Prepare cell lysate of alveolar-like organoids
4. Add 1 mL ice-cold PBS to the organoids.

SECTION: Prepare cell lysate of alveolar-like organoids
5. on ice Transfer organoids to 15 mL LoBind Tube (with 1000 µL pipette).

SECTION: Prepare cell lysate of alveolar-like organoids
6. Centrifuge 300 x g, 5 min, 4 °C.

SECTION: Prepare cell lysate of alveolar-like organoids
7. on ice Remove supernatant carefully.

SECTION: Prepare cell lysate of alveolar-like organoids
8. on ice Make sure the protease inhibitor is added to the RIPA buffer.

SECTION: Prepare cell lysate of alveolar-like organoids
9. Add 75 µL RIPA buffer + Protease inhibitor to pellet.

SECTION: Prepare cell lysate of alveolar-like organoids
10. Breake organoid pellet by repeated resuspension (3 times) using a disposable syringe with 27G needle.
SECTION: Prepare cell lysate of alveolar-like organoids
11. Transfer sample to 1.5 mL LoBind tube.

SECTION: Prepare cell lysate of alveolar-like organoids
12. Centrifuge 16000 x g, 10 min, 4 °C.

SECTION: Prepare cell lysate of alveolar-like organoids
13. on ice Collect and transfer supernatant to 1.5 mL LoBind tube (~50 µL).

SECTION: Prepare cell lysate of alveolar-like organoids
14. Freeze at -80 °C or continue.

SECTION: Protein quantification and preparation before Western blot
15. 
Use 5 µL of RIPA-lysate in DC Assay (a dilution of the sample might be required, 1:10 or 1:50 in PBS, follow manufacturer's instructions) for protein quantification.

SECTION: Protein quantification and preparation before Western blot
16. Prepare appropriate amount of sample by using 50 µg protein lysate according to DC assay results with 4x Laemmli protein sample buffer for SDS-PAGE.

SECTION: Protein quantification and preparation before Western blot
17. Shake for 7 min at 95 °C (1000 rpm).

SECTION: SDS-PAGE
18. Commercial gel used: Bio Rad 7.5% (Mini-Protean TGX Precast Gels Cat# 456-1023).
SECTION: SDS-PAGE
19. Arrange gel chambers inside the electrophorator and fill the tank with 1x Running-Buffer (fill the chamber formed by the gels with new 1x Running-Buffer, check if everything is tight and fill the tank with 1x Running-Buffer until it is half full). Remove the comb carefully.
SECTION: SDS-PAGE
20. Load 5 µL Kaleidoscope Protein Standard in lane 1 (Precision Plus Protein Kaleidoscope Prestained Protein Standards #1610375).

SECTION: SDS-PAGE
21. Add 50 µg sample/lane (can be increased to 100 µg sample/lane for low expressed proteins).

SECTION: SDS-PAGE
22. Run the gel for up to 90 min at 100 V (until the blue front line runs out of the gel).

SECTION: Blotting
23. After the SDS-Page is done, do not switch off the power (due to diffusing proteins) until everything is prepared for blotting. It is better to reduce the voltage instead.
SECTION: Blotting
24. Before use keep bottle of 1x blotting buffer in the freezer for 90 min. Equilibrate PVDF membrane (0.45 micromolar (µM) methanol for 15 s) and transfer it to the 1x blotting buffer. Add sponges and filter papers in 1x blotting buffer. For each gel, you need 2 sponges and 4 filter papers. 
SECTION: Blotting
25. Carefully open gel chamber and equilibrate gel in 1x blotting buffer for 2 min.


SECTION: Blotting
26. Create a transfer sandwich as follows:

White side of retainer (top)
Sponge
2 filter papers
PVDF Membrane
SDS Gel
2 filter papers
Sponge
Black side of retainer (bottom)
SECTION: Blotting
27. Check out that there are no air bubbles between the gel and the membrane. Use a pipette to squeeze out extra liquid and bubbles.
SECTION: Blotting
28. Relocate the sandwich to the transfer apparatus. Add ice and fill with 1x Blotting-Buffer until the sandwich is completely covered.
SECTION: Blotting
29. Transfer for 60 min at 100 V.

SECTION: Blocking and antibody incubation
30. Wash membrane with PBS.
SECTION: Blocking and antibody incubation
31. Block membrane in Odyssey blocking solution for 60 min.

SECTION: Blocking and antibody incubation
32. Cut membrane for multiple protein detection.
SECTION: Blocking and antibody incubation
33. Add primary antibody (diluted in Odyssey blocking solution pure) and incubate membrane on a shaker overnight at 4 °C.

SECTION: Blocking and antibody incubation
34. Wash membrane thrice with 1x PBS-T for 5 min each.

SECTION: Blocking and antibody incubation
35. Incubate membrane for 60 min at room temperature with secondary antibody solution (in 1x PBS-T with 5% milk).



SECTION: Blocking and antibody incubation
36. Wash membrane twice with 1x PBS-T for 5 mineach.
SECTION: Blocking and antibody incubation
37. Wash membrane with 1x PBS for 5 min.

SECTION: Blocking and antibody incubation
38. Prepare ECL solution and incubate on membrane according to manufactures instructions (2 min for Thermo Scientific Kit and 5 min for Amersham Kit). The more sensitive Amersham Kit is used for ACE2 detection, all other proteins are detected using the Thermo Scientific Kit.

SECTION: Blocking and antibody incubation
39. Expose film until an adequate signal can be determined