Fluorescent Western Protocol

Lynn Doran, Steven J Burgess

Published: 2022-01-13 DOI: 10.17504/protocols.io.b3q6qmze

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:

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.

Steps

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 0h 2m 0s min

Rinse membrane with dH₂O

Discard PBS and incubate with 1h 0m 0s 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.

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

Incubate with primary antibody (appropriate dilution in at 4°C (in cold room) with gentle agitation on a platform shaker 50rpm .

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 50rpm. Repeat 3 times.

Note

If high background signal is observed, increase wash time to 20 minutes.

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.

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.

10.

Incubate for 1h 0m 0s Room temperature with gentle agitation on a platform shaker.

Note

Incubation longer than one hour will lead to high background signal.

11.

Pour off the secondary antibody and rinse membrane with distilled water to remove residual blocking agent.

12.

Cover the membrane with PBS-T, agitate 80rpm

Note

If high background signal is observed, increase wash time to 20 minutes.

13.

Discard PBS-T. Repeat step 12 three times.

Note

More washes (x5) and for longer can be done to reduce background

14.

Rinse then cover the membrane with 1x PBS, 80rpm.

15.

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

Equipment

Value	Label
Odyssey CLx	NAME
Imaging System	TYPE
LI-COR	BRAND
Odyssey CLx	SKU

Note

Ensure that the platform of the Odyssey CLx is thoroughly cleaned of residual protein from previous blots using isopropanol.

Fluorescent Western Protocol

Abstract

Before start

Steps

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