TITLE: Western Blots with S. rosetta lysate (version 1)
AUTHORS: Fredrick leon

[DESCRIPTION]
This protocol utilizes the preferential lysis of S. rosetta to obtain choanoflagellate-specific proteins from co-culture with feeder bacteria. Protein concentrations are normalized by total protein stain, and the use of LICOR blocking buffer offers increased specificity of antibodies. Additionally, robust detection of specific proteins can be achieved when combined with genome editing of epitope tags into proteins of interest. This protocol was developed with essential input from Alain Garcia de las Bayonas and Flora Rutaganira.

[STEPS]
SECTION: Prepare lysis buffer
1. The following recipe and protocol is based entirely on the preferential lysis of S. rosetta. Modifications have been made to remove all nucleic acids, and to remove unnecessary components. 
 
 
SECTION: Count, Harvest, and Lyse cells
2. Follow the lysis steps of the preferential lysis protocol. 
SECTION: Determine protein concentration
3. Using the Bradford assay kit, prepare the BSA standard curves by diluting the standards into fresh lysis buffer following the schemebelow. The detailed protocol can be found here. I recommend using the "standard microplate protocol" found in the linked pdf.  
 



  

SECTION: Determine protein concentration
4. Pipette 10 µL of each standard or unknown sample into clear bottom 96-well plates  .

SECTION: Determine protein concentration
5. Add 300 µL of the Assay Reagent to each well, mix by gently taping on all sides for 60 seconds.
SECTION: Determine protein concentration
6. Incubate plate for 10 minutes at RT.
SECTION: Determine protein concentration
7. Measure the absorbance at or near 595 nm on a plate reader.
SECTION: Determine protein concentration
8. Subtract the average blank 595 nm measurement from the standards and sample values.
SECTION: Determine protein concentration
9. Use the standard curve to determine the protein concentration of each sample.
SECTION: Run SDS-PAGE
10. Aliquot 20 μg of total protein per sample (variable volume based off concentration) into pcr tubes, and denatured with 4x Laemmli SDS loading buffer (diluted to 1x).
SECTION: Run SDS-PAGE
11. Incubate denatured samples for 10 min at Room temperature if your proteins of interest are membrane proteins, or at 80 °C otherwise. Membrane proteins can crash out at higher temperatures.

SECTION: Run SDS-PAGE
12. Prepare your gel and running buffer. For smaller membrane proteins (25-100 kD), 10% tris-glycine gels are a good starting point.    
 
 

SECTION: Run SDS-PAGE
13. Load denatured protein samples into the gel. Run according to manufacturers' guidelines.  
SECTION: Prepare the transfer stack
15. While the gel from the previous step runs, prepare the transfer buffer: 
3.02 g Tris base (25 mM final); 14.4 g glycine (192mM final); 100 ml methanol (10% final), brought to 1L with ice cold water. 
SECTION: Prepare the transfer stack
14. In advance, prepare 3.5" x 3.5" squares of filter paper and transfer membrane. (0.45 µM nitrocellulose membranes are a good starting point) 
SECTION: Prepare the transfer stack
16. Once the gel is done running, prepare the transfer stack. Find a shallow container to place the transfer stack cassette inside. The container should be shallow and wide enough for the cassette to lay open and foryour hands to access it easily.
SECTION: Prepare the transfer stack
17.  Place foam/fiber pads on both sides of the cassette. Then pour in the transfer buffer until it covers the foam/fiber pads.
SECTION: Prepare the transfer stack
18. On one side of the cassette, use clean tweezers and place 2 squares of filter paper. Ensure they're submerged in the transfer buffer. Use the side of a clean P1000 pipette tip to gently rub and remove bubbles from between the filter papers. 
 

 

SECTION: Prepare the transfer stack
19. Take the gel and crack open the plastic housing. Remove the lanes at the top and the thicker section at the bottom (so the gel will lay flat) with the edge of the gel knife, while leaving the gel adhered to one-half of the plastic housing. Place a third filter paper on top of the gel and gently push the filter paper onto the gel until it sticks. Gently pull back the filter paper while ensuring the gel separates with the paper. 
 

SECTION: Prepare the transfer stack
20. Slowly place the filter paper adhered gel onto the previously submerged filter papers, with the gel facing UP. At this stage there will be plenty of bubbles under the gel. Repeat the bubble removing step slowly until no more bubbles are visible. 
 
 
SECTION: Prepare the transfer stack
21. Using tweezers, take a square of transfer membrane, and cut off a small corner of the gel. Match the cut corner of the membrane to the cut corner of the gel. While ensuring the gel is submerged or at least recently wetted with buffer, precisely place the membrane over the gel. Lay one edge of the membrane down first and slowly lower the rest of the membrane. Remove bubbles as before.
 
 
SECTION: Prepare the transfer stack
22. Place 3 more filter papers on top, removing bubbles as before. 
SECTION: Prepare the transfer stack
23. Close the cassette. This is a diagram of how the stack will look once closed. 
 

SECTION: Transfer
24. Pour any remaining transfer buffer into the transfer tank. Then place the loaded cassette into the tank. Pour the leftover buffer from the container you assembled the cassette in. 
SECTION: Transfer
25. Put the tank inside a secondary container and surround with ice. Bring the entire set up into a cold room. 
SECTION: Transfer
26. Run the transfer at 50 V for 30 minutes, then 100 V for 60 minutes
SECTION: Total protein stain
27. Total protein stains are done with the LICOR 700 total protein stain kit. A detailed protocol can be found here.

  

SECTION: Blocking
35. Take the imaged membrane, and place in a small container for blocking. Block in LICOR Intercept Blocking Buffer, using enough to cover the membrane completely. Incubate for 60 min at 37 °C with gently shaking
  

SECTION: Immunodetection
39. After incubation, decant the blocking buffer and wash the membrane in TBST or PBST (1x TBS or PBS plus 0.2% Tween-20) for 5 min at Room temperature  with gently shaking, repeat two more times for a total of three washes. 

SECTION: Total protein stain
28. Take the membrane from the transfer stack, and place in a clean container. Dry for10 min at 37 °C 
 


SECTION: Total protein stain
29. incubate the membrane in TBS or PBS (no detergent) for 5 min  at Room temperature with gentle shaking

SECTION: Total protein stain
30. Wash the membrane in water
SECTION: Total protein stain
31. Incubate the membrane in 5 mL for 5 min  at Room temperature with gentle shaking. Decant afterwards.

SECTION: Total protein stain
32. Wash with 5 mL for 30 s at Room temperature with gentle shaking twice, decanting in between. 

SECTION: Total protein stain
33. Wash the membrane in water, and keep the membrane in water while preparing to image.
SECTION: Total protein stain
34. Immediately image the membrane in the 700 nm channel on a membrane imager. 
SECTION: Blocking
36. Decant the blocking buffer, and replace with fresh blocking buffer.  Incubate for 60 min at 4 °C with gently shaking
SECTION: Immunodetection
37. The following day, decant the blocking buffer, and add back a minimal volume of blocking buffer that covers the membrane. This should be done with a serological pipette so you know the final volume.  
SECTION: Immunodetection
38. In the blocking buffer, dilute your antibodies. Incubate for 120 min at Room temperature with gentle shaking.
 



SECTION: Immunodetection
40. After the third wash, image the membrane in the 800 nm channel on a membrane imager.