Sep 13, 2023
Version 2
Fluorescent Western Protocol V.2
Version 1 is forked from Fluorescent Western Protocol
This protocol is a draft, published without a DOI.
- 1Realizing Increased Photosynthetic Efficiency (RIPE);
- 2University of Illinois at Urbana-Champaign
- UIUC Long Lab
Protocol Citation: Lynn Doran, Steven J Burgess 2023. Fluorescent Western Protocol. protocols.io https://dx.doi.org/Version created by Lynn Doran
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: September 13, 2023
Last Modified: September 13, 2023
Protocol Integer ID: 87732
Keywords: Western Blot, Fluorescent Western Protocol, Protein Analysis, fluorescent western blot detection, fluorescent western protocol analysis of protein, fluorescent immunoblot with optional total protein stain, using fluorescent immunoblot, fluorescent western protocol analysis, secondary antibody, primary antibody, antibody, optional total protein stain, choice of secondary antibody, choice of primary antibody, chlorophyll autofluorescence, licor, problems with chlorophyll autofluorescence, good westerns gone, protein
Abstract
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:
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.
Materials
Reagents
- Intercept® T20 (PBS) Antibody DiluentLI-CORCatalog #927-75001
- Instant Nonfat Dry Milk
- Methanol
- Ultrapure water.
Materials
- Graduated cylinder
- Reagent bottle
- Micropipette, 1000 ul
- Micropipette, 10 ul
- Pipette tips, 100-1000 ul
- Pipette tips, 1-10 ul
Equipment
- Odyssey CLx Imager (LI-COR Biosciences)
- Shaking platform
- Incubator, capable of 37C.
Protocol materials
Intercept® T20 (PBS) Antibody DiluentLI-CORCatalog #927-75001
Intercept (PBS) Blocking BufferLI-CORCatalog #927-70001
Before start
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.
Note
Boxes should be thoroughly cleaned between uses. Residual protein contamination from previous blots can lead to high background signal. If background signal is increasing, clean boxes with 70% ethanol and dry thoroughly between uses.
Optional: Revert 700 Total Protein Stain
2h 2m
Add methanol to the Revert 700 Total Protein Stain reagents as indicated on each bottle.
Rinse the transfer buffer from the membrane after transblot using ultrapure water.
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.
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.
Rinse membrane with ultrapure water.
Stain membrane with 5 mL of Revert 700 Total Protein Stain.
Measure the volume with a graduated cylinder to save expensive reagents.
Incubate with shaking for 5 minutes at room temperature.
Discard stain in appropriate hazardous waste.
Add 5 mL of Revert 700 Wash Solution.
Measure the volume with a graduated cylinder to save expensive reagents.
Incubate with shaking 30 seconds at room temperature.
Discard wash in appropriate hazardous waste.
Repeat 5 mL Revert 700 Wash.
Discard wash solution and rinse membrane with ultrapure water.
Note
This optional protocol is for total protein quantification for a single-color western blot on the 800 nm channel only. If a two-color western blot on both 700 and 800 nm channel is planned, refer to the Revert 700 Total Protein Stain Insert for the appropriate destain procedure.
Image on 700 nm channel on Odyssey Imaging System.
Note
Fluorescent Western Protocol
Wet with 1x PBS for 00:02:00 min
2m
Rinse membrane with dH2O
Discard PBS and incubate with Intercept (PBS) Blocking BufferLI-CORCatalog #927-70001 01:00:00 at Room temperature
Note
Blocking prevents unspecific binding of antibody and lowers background signal.
As an alternative to Intercept Blocking Buffer you can use PBS 5% w/v milk powder (no Tween-20). Typical mini-blots use ~15 mL blocking buffer (15 mL 1X PBS, 0.75 g non-fat dry milk). Do not use milk powder as a blocking agent if protein of interest is phosphorylated.
1h
Prepare 1 L PBS-T solution by diluting 100 mL of 10X PBS
Incubate with primary antibody (appropriate dilution in Intercept® T20 (PBS) Antibody DiluentLI-CORCatalog #927-75001 at 4 °C Overnight (in cold room) with gentle agitation on a platform shaker 50 rpm, 4°C .
Note
As an alternative to Intercept Antibody Diluent you can use PBST (0.1% Tween-20, 5% milk). Typical mini-blots use ~15 mL blocking buffer (15 mL 1X PBST, 0.75 g non-fat dry milk).
1:2,000 to 1:5,000 are common dilutions for primary antibodies. The ideal dilution for the primary antibody will vary based on sample type and antibody binding capacity and must be determined empirically.
Pour off the primary antibody and rinse the membrane with PBS-T.
Note
Some primary antibodies can be re-used multiple times depending on the concentration used, in this instance collect the primary antibody in a tube and store the solution at -20 oC before re-use.
Cover the membrane with PBS-T, shake vigorously on a platform shaker at 50 rpm, Room temperature , 00:10:00 . Repeat 3 times.
Note
If high background signal is observed, increase wash time to 20 minutes.
Create a working dilution of secondary antibody using Intercept® T20 (PBS) Antibody DiluentLI-CORCatalog #927-75001. . 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.
Note
As an alternative to Intercept Antibody Diluent you can use PBST (0.1% Tween-20, 5% milk). Typical mini-blots use ~15 mL blocking buffer (15 mL 1X PBST, 0.75 g non-fat dry milk).
1:20,000 is a common dilution for secondary antibodies. Consult manufacturer's recommendations and the ideal dilution for the secondary antibody will vary based on sample type and antibody binding capacity and may need to be determined empirically.
Note
Ensure that secondary antibody is compatible with primary antibody. For plants, an antibody that fluoresces at 800 nm is recommended. Chlorophyll fluoresces near 700 nm and may cause high background signal or interfere with target signal.
Incubate for 01:00:00 Room temperature with gentle agitation on a platform shaker.
Note
Incubation longer than one hour can lead to high background signal.
1h
Pour off the secondary antibody and rinse membrane with distilled water to remove residual blocking agent.
Cover the membrane with PBS-T, agitate 80 rpm, Room temperature , 00:10:00
Note
If high background signal is observed, increase wash time to 20 minutes.
Discard PBS-T. Repeat step 12 three times.
Note
More washes (x5) and for longer can be done to reduce background
Rinse then cover the membrane with 1x PBS, 80 rpm, Room temperature , 00:10:00 .
Proceed to imaging blot on LI-COR Odyssey CLx imaging system.
Equipment
Odyssey CLx
NAME
Imaging System
TYPE
LI-COR
BRAND
Odyssey CLx
SKU
LINK
Note
Ensure that the platform of the Odyssey CLx is thoroughly cleaned of residual protein from previous blots using isopropanol.