TITLE: Fluorescent Western Protocol
AUTHORS: Lynn Doran, Steven J Burgess

[DESCRIPTION]
Analysis of proteins using fluorescent immunoblot with optional total protein stain.

Note:  
- The choice of secondary antibody depends on the choice of primary antibody, whether it is derived from a mouse (monoclonal) or a rabbit (polyclonal).  
- It is advisable to stick to the 800CW wavelength to avoid problems with chlorophyll autofluorescence encountered with the 680CW antibodies.  

Literature: 
Licor's "Fluorescent Western Blot Detection"
Licor's "Good Westerns Gone Bad"

[BEFORE_START]
Isolate total protein via Leaf Protein Extraction for Immunoblot (Soybean, Cowpea, Tobacco).
Quantify protein via Protein Concentration Determination using Qubit 4 Fluorometer.
Separate protein components via SDS-PAGE gel electrophoresis.
Transfer protein to a membrane via Protein Transfer using Bio-rad TransBlot Turbo.

[GUIDELINES]
Do not touch the membrane.  Always use tweezers to move the membrane to avoid protein contamination from your hands.  Always use clean materials to handle and store the membranes as residual protein from dirty materials can cause high background on the membranes.

[STEPS]
1. 
Keep membranes in the black Western Blot incubation box for all steps, this is important after adding the secondary antibody because the signal is light-sensitive and will become bleached if exposed to light for a long enough period.

 


SECTION: Fluorescent Western Protocol
16. Wet with 1x PBS for 2 min min

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17. Rinse membrane with dH2O
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18. Discard PBS and incubate with   60 min  at Room temperature
 



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20. Incubate with primary antibody (appropriate dilution in    at 4 °C    (in cold room) with gentle agitation on a platform shaker   .
 


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19. Prepare 1 L PBS-T solution by diluting 100 mL of 10X PBS

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21. Pour off the primary antibody and rinse the membrane with PBS-T.
 

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22. Cover the membrane with PBS-T, shake vigorously on a platform shaker at  . Repeat 3 times.

 


SECTION: Fluorescent Western Protocol
23. Create a working dilution of secondary antibody using . . For PVDF membranes only, add 0.01% SDS to the antibody diluent.    Microcentrifuge secondary antibody and pipette from supernatant to precipitate out any protein complexes that may have formed during storage.  

 

 

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24. Incubate for 60 min Room temperature with gentle agitation on a platform shaker.
 

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25. Pour off the secondary antibody and rinse membrane with distilled water to remove residual blocking agent.
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26. Cover the membrane with PBS-T, agitate  
 

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27. Discard PBS-T. Repeat step 12  three times.
 

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28. Rinse then cover the membrane with 1x PBS,  . 

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29. Proceed to imaging blot on LI-COR Odyssey CLx imaging system. 
 

 

SECTION: Optional: Revert 700 Total Protein Stain
2. Add methanol to the Revert 700 Total Protein Stain reagents as indicated on each bottle.
SECTION: Optional: Revert 700 Total Protein Stain
3. Rinse the transfer buffer from the membrane after transblot using ultrapure water.
SECTION: Optional: Revert 700 Total Protein Stain
4. Place the membrane on a Whatman 1 filter paper or drape it across the seam of the incubation box and dry at 37C for 10 minutes.

Alternatively, it can be dried overnight at room temperature if short on time on day 1.
SECTION: Optional: Revert 700 Total Protein Stain
5. Rehydrate the membrane for 5 minutes.
Nitrocellulose: rehydrate in PBS for 5 minutes with shaking.
PVDF: Rehydrate in 100% methanol for 30 seconds, then in PBS for 5 min with shaking.
SECTION: Optional: Revert 700 Total Protein Stain
6. Rinse membrane with ultrapure water.
SECTION: Optional: Revert 700 Total Protein Stain
7. Stain membrane with 5 mL of Revert 700 Total Protein Stain.

Measure the volume with a graduated cylinder to save expensive reagents.
SECTION: Optional: Revert 700 Total Protein Stain
8. Incubate with shaking for 5 minutes at room temperature.
SECTION: Optional: Revert 700 Total Protein Stain
9. Discard stain in appropriate hazardous waste.
SECTION: Optional: Revert 700 Total Protein Stain
10. Add 5 mL of Revert 700 Wash Solution.  
Measure the volume with a graduated cylinder to save expensive reagents.
SECTION: Optional: Revert 700 Total Protein Stain
12. Discard wash in appropriate hazardous waste.
SECTION: Optional: Revert 700 Total Protein Stain
13. Repeat 5 mL Revert 700 Wash.
SECTION: Optional: Revert 700 Total Protein Stain
11. Incubate with shaking 30 seconds at room temperature.
SECTION: Optional: Revert 700 Total Protein Stain
14. Discard wash solution and rinse membrane with ultrapure water.

 

SECTION: Optional: Revert 700 Total Protein Stain
15. Image on 700 nm channel on Odyssey Imaging System.