Hints and tips for fluorescent western blotting

Get started with fluorescent western blot with these quick tips on choosing your secondary and protocol optimization.

Choosing your secondary

For clear and bright bands use an antibody that has been optimized for fluorescent western blot

  • When multiplexing, ensure that each individual primary antibody used is from a different species.
  • Use secondary antibodies which are highly cross-adsorbed to minimize cross-species reactivity.
  •  IRDye® secondary antibodies have been tested for over 600 different protein targets and are fully optimized for fluorescent western blot.

IRDye ® 680RD

Protein with higher expression (eg Housekeeping gene)

Excitation: 679 nm

Emission: 696 nm

700 channel

Dilution range:

1:5,000 - 1:25,000

IRDye ® 800CW

Protein lower expression (eg normally the protein of interest)

Excitation: 778 nm

Emission: 795 nm

800nm channel

Dilution range:

1:5,000 - 1:25,000

    Optimizing your antibody 

    Get better signal-to-background ratio

    • Find the optimal antibody concentration by individually titrating the primary and secondary antibodies. Test several dilutions and select the one that yields the highest signal-to-background ratio.
    • Optimize conditions for individual antibody pairs (primary + conjugated secondary) separately, before attempting multi-color analysis.

    Steps and reagents to pay attention in your protocol

    • Membrances have a tendency to autofluoresce resulting in high background.  Choose specialist membranes which have been engineered to overcome this issue - low fluorescence western membrane (PVDF)
    • Undissolved particles within buffers (milk powder in blocking buffer for example) potentially can settle on the membrane and create fluorescent artifacts.  Therefore, we suggest using high quality reagents, allowing suitable time for all components to fully dissolve, and filter sterilize all buffers.
    • Handle the membrane with care, use blunt forceps and avoid scratching/creasing to prevent fluorescent artifacts.
    • Ink fluoresces, so mark the membrane with a pencil rather than pen.
    • Bromophenol blue itself fluoresces - so either run the dye front off the gel, or cut the gel part off, which contains the dye, prior to transfer.
    • Protect membrane from light during incubation and wash steps with aluminium foil.

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