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Fluorescent Western Blot Detection
Fluorescent western botting 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.
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.
How to find a secondary antibody
Step 1: Search for the target species of interest (e.g., “anti-mouse” or “goat anti-rabbit” or “alexa fluor 488”).
Step 2: Narrow results by host species, conjugate, application, and other criteria using side filters.
Learn more about the properties of individual fluorescent dyes
Use the Fluorescence SpectraViewer to help plan your experiments
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 |
|
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.
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.
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.
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.
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.
How to find a secondary antibody
Step 1: Search for the target species of interest (e.g., “anti-mouse” or “goat anti-rabbit” or “alexa fluor 488”).
Step 2: Narrow results by host species, conjugate, application, and other criteria using side filters.
Learn more about the properties of individual fluorescent dyes
Use the Fluorescence SpectraViewer to help plan your experiments
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 |
|
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.
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.
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.
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.
Watch this webinar to learn more about fluorescent western blotting
Webinar: Light up your western blots – fluorescent western blotting tips, tricks & more
Speaker: Paul Haney, Ph.D., Senior Product Manager, Protein and Cell Analysis, Thermo Fisher Scientific
Resources
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