Jan 13, 2022
Version 2
Fluorescent Western Protocol V.2
- 1University of Illinois at Urbana-Champaign;
- 2Realizing Increased Photosynthetic Efficiency (RIPE)
- GEGC lab UIUC
Protocol Citation: Steven J Burgess, Lynn Doran 2022. Fluorescent Western Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.b3q6qmzeVersion 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: January 13, 2022
Last Modified: July 12, 2023
Protocol Integer ID: 56830
Keywords: Western Blot, Fluorescent Western Protocol, Protein Analysis
Funders Acknowledgements:
Realizing Increased Photosynthetic Efficiency (RIPE) that is funded by the Bill & Melinda Gates Foundation, Foundation for Food and Agriculture Research, and the U.K. Foreign, Commonwealth & Development Office
Grant ID: OPP1172157
Abstract
Analysis of proteins using fluorescent immunoblot.
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:
Materials
Reagents
- Intercept® T20 (PBS) Antibody DiluentLicorCatalog #927-75001
- Instant Nonfat Dry Milk
- Methanol
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
Protocol materials
Intercept® T20 (PBS) Antibody DiluentLI-CORCatalog #927-75001
Intercept (PBS) Blocking BufferLI-CORCatalog #927-70001
Intercept® T20 (PBS) Antibody DiluentLI-CORCatalog #927-75001
Intercept® T20 (PBS) Antibody DiluentLI-CORCatalog #927-75001
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 not 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.
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.
Incubate for 01:00:00 Room temperature with gentle agitation on a platform shaker.
Note
Incubation longer than one hour will 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.