Fluorescent Western Blot Detection

Fluorescent western blotting provides accurate, quantitative, stable signals, and the ability to clearly evaluate multiple protein targets on a single blot. We offer optimized reagents, buffers and fluorescent conjugated antibodies so you can get the most from your fluorescent western blots.

Get more from your multiplex Westerns

Fluorescent western blot optimized reagents

To limit background fluorescence, specific fluorescent western blotting reagents have been developed and optimized to help you obtain the best sensitivity when using fluorescent conjugated-antibodies or probes. From sample preparation to protein detection these reagents and tools can help you limit background fluorescence.

Sample buffers for SDS-PAGE

Sample buffers containing bromophenol blue and certain other dyes will fluoresce and can contribute to increased background fluorescence. Care must be taken when using these sample buffers as majority of camera systems will optimize exposure to the bright dye front. We recommended using fluorescence-friendly sample buffers without bromophenol blue.

Thermo Fisher Scientific product Description Order
Fluorescent compatible sample buffer Fluorescent friendly sample buffer formulated without bromophenol blue or other dyes that interfere in the fluorescent channels but still providing a visible dye front. The buffer is ready-to-use for non-reducing SDS-PAGE or add reducing agent for reducing SDS-PAGE. LC2570

Molecular weight markers for fluorescent detection

Consideration needs to be taken when picking a molecular weight marker for fluorescent detection, to not overwhelm the fluorescent of your target proteins. We offer several protein ladders specifically designed for fluorescent western blotting.

 
  iBright Prestained Protein Ladder MagicMark XP Western Protein Standard PageRuler Prestained NIR Protein Ladder
Applications Fluorescence, chemiluminescent, direct visualization NIR fluorescence, chemiluminescent, chromogenic NIR fluorescence, direct visualization
Molecular weight range 11-250 kDa 20-220 kDa 11-250 kDa
Number of bands 12 9 10
No. of colors 10 single colored bands (prestained) unstained 10 single colored bands (prestained)
Major features Two unstained proteins (30 and 80 kDa) with IgG binding sites for chemiluminescent or fluorescent detection 9 proteins with IgG binding sites for chemiluminescent or fluorescent detection Reference band at 55kDa with greater intensity
Fluorescence wavelength NIR and determined by 2° antibodies used Determined by 2° antibodies used (only compatible with NIR dyes) NIR
Order LC5615 LC5602 26635

Membranes

To eliminate a major source of background fluorescence, we recommend membranes with low auto-fluorescence, which include nitrocellulose and specialty low-fluorescence PVDF membranes. We have several membranes that provide low background fluorescent for your experimental needs.

Thermo Fisher Scientific product Description Order
Low-Fluorescence PVDF 0.2 µm 7 cm x 8.4 cm (22860)
Nitrocellulose 0.45 µm 30cm x 3.5m (88018)
8cm x 12cm (77010)
8cm x 8cm (88025)
7.9cm x 10.5cm (88014)
Nitrocellulose 0.2 µm 8cm x 12cm (77012)
8cm x 8cm (88024)
7.9cm x 10.5cm (88013)

See our Membranes page for a complete list of all our available membrane

Blocking buffers

When performing fluorescent western blotting it is recommended to use only high-quality filtered buffers. Particles and contaminants in wash and blocking buffers can settle on membranes and create fluorescent artifacts. In addition, limit the use of detergents during blocking steps, as common detergents can auto-fluoresce and increase nonspecific background. Protein interactions are unpredictable; blocking buffers that work well in one system may result in high background in another. We recommend testing multiple blocking buffers to find the best signal-to-noise ratio.

  Thermo Fisher Scientific product Description Order
Start with Blocker FL Blocking Buffer Designed to reduce cross-reactivity and produce high signal-to-noise ratios 37565
Next StartingBlock Blocking Buffer Single purified protein, fast-blocking, broad applicability, available in PBS and TBS with and without Tween buffer; compatible with most antibodies as well as biotin-binding reagents PBS (37538)
TBS (37542)
Specialty SEA BLOCK Blocking Buffer Fish serum, less likely to have nonspecific binding with antibodies and other mammalian proteins 37527

Stripping buffer

Reprobing a stripped blot saves time and cost when samples are in limited quantities, when the same sample requires analysis by different antibodies, or when optimization is required. Traditional stripping buffers may only be effective for removing only low-affinity antibodies, leaving behind dye-labeled secondary antibodies that can overwelm the target signal when re-probing. Restore Fluorescent Western Blot stripping buffer was developed to assist in removing near-infrared (IR) dye-labeled secondary antibodies (680-800 nm) from western blots.

Thermo Fisher Scientific product Description Order
Restore Fluorescent Western Blot Stripping Buffer A gentle and highly effective reagent for quickly removing primary and near-infrared (IR) dye-labeled secondary antibodies from western blots transferred on PVDF membranes 62300

We offer a wide selection of fluorescent-dye conjugated secondary antibodies for your research, with over 60 different fluorescent dyes and dye combinations, and nanocrystals in a wide range specific target and host species.


Alexa Fluor Plus secondary antibodies

The growing demand for multiplex western blotting has driven the development of many new fluorescent dyes- such as Alexa Fluor Plus conjugates. These new fluorophores are brighter and more photo stable than the traditional fluorescein and rhodamine molecules traditionally used and comprise a broader range of non-overlapping spectra. Together with the advances in the digital imaging equipment these new fluorophores enable extremely powerful analyses in western blotting.

Features of Alexa Fluor Plus secondary antibodies

  • Higher signal-to-noise ratio, enabling detection of low-abundance targets
  • Up to 5.8 times higher signal to noise in western fluorescent blotting
  • Enhanced sensitivity and greater range of linear detection
  Invitrogen Alexa Fluor Plus Secondary Antibody products
Click on the catalog number below to choose.
Goat anti-Rabbit Goat anti-Mouse Goat anti-Chicken Donkey anti-Goat Donkey anti-Rabbit Donkey anti-Mouse
Alexa Fluor Plus 488 A32731 A32723 A32931 A32814 A32790 A32766
Alexa Fluor Plus 555 A32732 A32727 A32932 A32816 A32794 A32773
Alexa Fluor Plus 594 A32740 A32742 A32759 A32758 A32754 A32744
Alexa Fluor Plus 647 A32733 A32728 A32933 A32849 A32795 A32787
Alexa Fluor Plus 680 A32734 A32729 A32934 A32860 A32802 A32788
Alexa Fluor Plus 800 A32735 A32730 A32935 A32930 A32808 A32789

Fluorescent western blotting

Western blotting is a powerful widely preformed protein detection application. With the introduction of advanced digital imaging instruments like the Invitrogen iBright FL1000 Imaging System and improvements in fluorescent conjugate technologies, fluorescent western blotting systems are growing in popularity. These advancements provide access to fluorescence detection with reduced costs and improved sensitivity. Overall, the western blotting procedure is similar between detection methods, with fluorescent western blotting providing specific benefits.

Attribute Fluorescent Western Blotting
Signal source Direct signal from fluorophore
Signal duration Extended (weeks to months)
Sensitivity Good with a large range of fluorophores available
Consistency High reproducibility between blots
Detection Requires imaging instruments with suitable light sources and filters
Quantitation Multiplexing with an internal control makes normalization simpler
Other considerations
  • Care is needed to avoid fluorescence contamination
  • Longer exposure times can produce background

To get the most from your fluorescent western blots, particular steps should be optimized to limit and avoid fluorescence contamination. Here are a few tips to take into account when performing fluorescent western blotting.

Proper choice of membranes

Choosing the right membrane is critical to achieving good signal-to-noise ratio in fluorescent western blotting. One major source of background fluorescence is the transfer membrane. When performing fluorescent western blotting particularly with fluorophores that have emission maximums around 488 nm, use membranes with low-auto fluorescence including nitrocellulose and specialty low-fluorescent PVDF membranes.

Common membranes

Antibody selection

One of the main advantages of fluorescent western blotting is the ability to visualize multiple protein targets in a single blot. With recent advantages in imaging systems, now blots can be performed with up to 4 different fluorophores. Picking the right set of secondary antibodies will make multiplexing easier. Here are a few tips to follow:

  • Use secondary antibodies that are highly cross-adsorbed against other species to avoid cross-reactivity.
    • Cross-adsorbed secondary antibodies are polyclonal antibodies that are manufactured with an additional purification step to filter out members that bind to off-target species of immunoglobulin (IgG). The process decreases species cross-reactivity and increases specificity.
  • Use primary antibodies from different host species to avoid cross-reactivity. Ideally use a combination of antibodies from two distantly related species such as rat and rabbit, avoiding combinations like mouse and rat or goat and sheep
  • Focus on western blot–validated primary antibodies.
  • Use primary antibodies directly conjugated to a fluorophore if species differentiation is not possible
    (e.g., loading controls, common protein targets)
  • Take advantage of your epitope-tagged proteins where directly conjugated antibodies are readily available
  • Select fluorophores with optically distinct spectra to avoid cross-channel fluorescence.
Examples of fluorophore combinations
Spectraviewer
Example, excitation and emission spectra of recommended Alexa Fluor and Alexa Fluor Plus dyes combination for 4-plex western bolt. Use the Fluorescence SpectraViewer to help plan your experiments and put in your instruments specific filters.
Example multiplexing blots

Example multiplexing blots.

Proper choice of buffers for fluorescence

Many blocking buffers can be used successfully for fluorescent western blotting (PBS, TBS, BSA, casein etc.). Particles and contaminants in wash and blocking buffers can settle on membranes and create fluorescent artifacts. We recommend using only high-quality filtered buffers.

  • Limit the use of detergents during blocking steps, as common detergents can auto-fluoresce and actually increase nonspecific background. After blocking, detergents can be used.
  • Filter sterilize if making buffers from scratch.
Suggested fluorescent friendly blocking buffers

Fluorescent western blot optimized reagents

To limit background fluorescence, specific fluorescent western blotting reagents have been developed and optimized to help you obtain the best sensitivity when using fluorescent conjugated-antibodies or probes. From sample preparation to protein detection these reagents and tools can help you limit background fluorescence.

Sample buffers for SDS-PAGE

Sample buffers containing bromophenol blue and certain other dyes will fluoresce and can contribute to increased background fluorescence. Care must be taken when using these sample buffers as majority of camera systems will optimize exposure to the bright dye front. We recommended using fluorescence-friendly sample buffers without bromophenol blue.

Thermo Fisher Scientific product Description Order
Fluorescent compatible sample buffer Fluorescent friendly sample buffer formulated without bromophenol blue or other dyes that interfere in the fluorescent channels but still providing a visible dye front. The buffer is ready-to-use for non-reducing SDS-PAGE or add reducing agent for reducing SDS-PAGE. LC2570

Molecular weight markers for fluorescent detection

Consideration needs to be taken when picking a molecular weight marker for fluorescent detection, to not overwhelm the fluorescent of your target proteins. We offer several protein ladders specifically designed for fluorescent western blotting.

 
  iBright Prestained Protein Ladder MagicMark XP Western Protein Standard PageRuler Prestained NIR Protein Ladder
Applications Fluorescence, chemiluminescent, direct visualization NIR fluorescence, chemiluminescent, chromogenic NIR fluorescence, direct visualization
Molecular weight range 11-250 kDa 20-220 kDa 11-250 kDa
Number of bands 12 9 10
No. of colors 10 single colored bands (prestained) unstained 10 single colored bands (prestained)
Major features Two unstained proteins (30 and 80 kDa) with IgG binding sites for chemiluminescent or fluorescent detection 9 proteins with IgG binding sites for chemiluminescent or fluorescent detection Reference band at 55kDa with greater intensity
Fluorescence wavelength NIR and determined by 2° antibodies used Determined by 2° antibodies used (only compatible with NIR dyes) NIR
Order LC5615 LC5602 26635

Membranes

To eliminate a major source of background fluorescence, we recommend membranes with low auto-fluorescence, which include nitrocellulose and specialty low-fluorescence PVDF membranes. We have several membranes that provide low background fluorescent for your experimental needs.

Thermo Fisher Scientific product Description Order
Low-Fluorescence PVDF 0.2 µm 7 cm x 8.4 cm (22860)
Nitrocellulose 0.45 µm 30cm x 3.5m (88018)
8cm x 12cm (77010)
8cm x 8cm (88025)
7.9cm x 10.5cm (88014)
Nitrocellulose 0.2 µm 8cm x 12cm (77012)
8cm x 8cm (88024)
7.9cm x 10.5cm (88013)

See our Membranes page for a complete list of all our available membrane

Blocking buffers

When performing fluorescent western blotting it is recommended to use only high-quality filtered buffers. Particles and contaminants in wash and blocking buffers can settle on membranes and create fluorescent artifacts. In addition, limit the use of detergents during blocking steps, as common detergents can auto-fluoresce and increase nonspecific background. Protein interactions are unpredictable; blocking buffers that work well in one system may result in high background in another. We recommend testing multiple blocking buffers to find the best signal-to-noise ratio.

  Thermo Fisher Scientific product Description Order
Start with Blocker FL Blocking Buffer Designed to reduce cross-reactivity and produce high signal-to-noise ratios 37565
Next StartingBlock Blocking Buffer Single purified protein, fast-blocking, broad applicability, available in PBS and TBS with and without Tween buffer; compatible with most antibodies as well as biotin-binding reagents PBS (37538)
TBS (37542)
Specialty SEA BLOCK Blocking Buffer Fish serum, less likely to have nonspecific binding with antibodies and other mammalian proteins 37527

Stripping buffer

Reprobing a stripped blot saves time and cost when samples are in limited quantities, when the same sample requires analysis by different antibodies, or when optimization is required. Traditional stripping buffers may only be effective for removing only low-affinity antibodies, leaving behind dye-labeled secondary antibodies that can overwelm the target signal when re-probing. Restore Fluorescent Western Blot stripping buffer was developed to assist in removing near-infrared (IR) dye-labeled secondary antibodies (680-800 nm) from western blots.

Thermo Fisher Scientific product Description Order
Restore Fluorescent Western Blot Stripping Buffer A gentle and highly effective reagent for quickly removing primary and near-infrared (IR) dye-labeled secondary antibodies from western blots transferred on PVDF membranes 62300

We offer a wide selection of fluorescent-dye conjugated secondary antibodies for your research, with over 60 different fluorescent dyes and dye combinations, and nanocrystals in a wide range specific target and host species.


Alexa Fluor Plus secondary antibodies

The growing demand for multiplex western blotting has driven the development of many new fluorescent dyes- such as Alexa Fluor Plus conjugates. These new fluorophores are brighter and more photo stable than the traditional fluorescein and rhodamine molecules traditionally used and comprise a broader range of non-overlapping spectra. Together with the advances in the digital imaging equipment these new fluorophores enable extremely powerful analyses in western blotting.

Features of Alexa Fluor Plus secondary antibodies

  • Higher signal-to-noise ratio, enabling detection of low-abundance targets
  • Up to 5.8 times higher signal to noise in western fluorescent blotting
  • Enhanced sensitivity and greater range of linear detection
  Invitrogen Alexa Fluor Plus Secondary Antibody products
Click on the catalog number below to choose.
Goat anti-Rabbit Goat anti-Mouse Goat anti-Chicken Donkey anti-Goat Donkey anti-Rabbit Donkey anti-Mouse
Alexa Fluor Plus 488 A32731 A32723 A32931 A32814 A32790 A32766
Alexa Fluor Plus 555 A32732 A32727 A32932 A32816 A32794 A32773
Alexa Fluor Plus 594 A32740 A32742 A32759 A32758 A32754 A32744
Alexa Fluor Plus 647 A32733 A32728 A32933 A32849 A32795 A32787
Alexa Fluor Plus 680 A32734 A32729 A32934 A32860 A32802 A32788
Alexa Fluor Plus 800 A32735 A32730 A32935 A32930 A32808 A32789

Fluorescent western blotting

Western blotting is a powerful widely preformed protein detection application. With the introduction of advanced digital imaging instruments like the Invitrogen iBright FL1000 Imaging System and improvements in fluorescent conjugate technologies, fluorescent western blotting systems are growing in popularity. These advancements provide access to fluorescence detection with reduced costs and improved sensitivity. Overall, the western blotting procedure is similar between detection methods, with fluorescent western blotting providing specific benefits.

Attribute Fluorescent Western Blotting
Signal source Direct signal from fluorophore
Signal duration Extended (weeks to months)
Sensitivity Good with a large range of fluorophores available
Consistency High reproducibility between blots
Detection Requires imaging instruments with suitable light sources and filters
Quantitation Multiplexing with an internal control makes normalization simpler
Other considerations
  • Care is needed to avoid fluorescence contamination
  • Longer exposure times can produce background

To get the most from your fluorescent western blots, particular steps should be optimized to limit and avoid fluorescence contamination. Here are a few tips to take into account when performing fluorescent western blotting.

Proper choice of membranes

Choosing the right membrane is critical to achieving good signal-to-noise ratio in fluorescent western blotting. One major source of background fluorescence is the transfer membrane. When performing fluorescent western blotting particularly with fluorophores that have emission maximums around 488 nm, use membranes with low-auto fluorescence including nitrocellulose and specialty low-fluorescent PVDF membranes.

Common membranes

Antibody selection

One of the main advantages of fluorescent western blotting is the ability to visualize multiple protein targets in a single blot. With recent advantages in imaging systems, now blots can be performed with up to 4 different fluorophores. Picking the right set of secondary antibodies will make multiplexing easier. Here are a few tips to follow:

  • Use secondary antibodies that are highly cross-adsorbed against other species to avoid cross-reactivity.
    • Cross-adsorbed secondary antibodies are polyclonal antibodies that are manufactured with an additional purification step to filter out members that bind to off-target species of immunoglobulin (IgG). The process decreases species cross-reactivity and increases specificity.
  • Use primary antibodies from different host species to avoid cross-reactivity. Ideally use a combination of antibodies from two distantly related species such as rat and rabbit, avoiding combinations like mouse and rat or goat and sheep
  • Focus on western blot–validated primary antibodies.
  • Use primary antibodies directly conjugated to a fluorophore if species differentiation is not possible
    (e.g., loading controls, common protein targets)
  • Take advantage of your epitope-tagged proteins where directly conjugated antibodies are readily available
  • Select fluorophores with optically distinct spectra to avoid cross-channel fluorescence.
Examples of fluorophore combinations
Spectraviewer
Example, excitation and emission spectra of recommended Alexa Fluor and Alexa Fluor Plus dyes combination for 4-plex western bolt. Use the Fluorescence SpectraViewer to help plan your experiments and put in your instruments specific filters.
Example multiplexing blots

Example multiplexing blots.

Proper choice of buffers for fluorescence

Many blocking buffers can be used successfully for fluorescent western blotting (PBS, TBS, BSA, casein etc.). Particles and contaminants in wash and blocking buffers can settle on membranes and create fluorescent artifacts. We recommend using only high-quality filtered buffers.

  • Limit the use of detergents during blocking steps, as common detergents can auto-fluoresce and actually increase nonspecific background. After blocking, detergents can be used.
  • Filter sterilize if making buffers from scratch.
Suggested fluorescent friendly blocking buffers