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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 to help you get the most from your fluorescent western blots.
Get more from your multiplex westerns Fluorescent secondary antibodies
Fluorescent western blot optimized reagents
To limit background fluorescence, specific fluorescent western blotting reagents have been developed and optimized to help you obtain excellent 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 can optimize exposure to the bright dye front. We recommended using fluorescence-friendly sample buffers without bromophenol blue.
| Thermo Fisher Scientific product | Description | Cat. No. |
|---|---|---|
| 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 | PageRuler Plus Prestained Protein Ladder | PageRuler Prestained NIR Protein Ladder | |
|---|---|---|---|
| Applications | Fluorescence, chemiluminescent, direct visualization | NIR and RGB fluorescence, direct visualization | NIR fluorescence, direct visualization |
| Molecular weight range | ~10-250 kDa | ~10-250 kDa | ~11-250 kDa |
| Number of bands | 12 | 9 | 10 |
| No. of colors | 10 single colored bands (prestained) | 9 colored bands- six blue, one green and two orange bands (prestained) | 10 single colored bands (prestained) |
| Major features | Two unstained proteins (30 and 80 kDa) with IgG binding sites for chemiluminescent or fluorescent detection | Three colors for quick reference of approximate size | Reference band at 55kDa with greater intensity |
| Fluorescence wavelength | NIR and determined by 2° antibodies used | 700 nm and 550nm | NIR |
| Cat. No. | LC5615 | 26619 | 26635 |
Membranes for fluorescent western blotting
To eliminate a major source of background fluorescence, use transfer membranes with low autofluorescence across all channels. While nitrocellulose membranes (e.g., Cat. No. 88018) typically exhibit lower background fluorescence than PVDF, low fluorescence PVDF membranes (e.g., Cat. No. 67800) perform well in all common fluorescent channels enabling flexible multiplexing strategies.
| Thermo Fisher Scientific product | Pore size | Format |
|---|---|---|
| Low fluorescence PVDF | 0.3 µm | Roll (8.3 cm x 10 m) Pre-cut sheets, mini (8.3 x 7.3 cm) Pre-cut sheets, midi (13.5 x 8.3 cm) Membrane/filter paper sandwiches, mini (8.3 x 7.3 cm) Membrane/filter paper sandwiches, midi (13.5 x 8.3 cm) |
| Nitrocellulose | 0.45 µm | Roll (30 cm x 3.5 m) Pre-cut sheets, mini (8 x 8 cm) Pre-cut sheets, midi (8 x 12 cm) |
| Nitrocellulose | 0.2 µm | Pre-cut sheets, mini (8 x 8 cm) Pre-cut sheets, midi (12 x 8 cm) |
Figure 1.Invitrogen Low Fluorescence PVDF exhibits lower autofluorescence when compared to nitrocellulose and standard PVDF membranes. Unprocessed membranes were hydrated and imaged together in six different fluorescent channels on a compatible digital western blot imaging system. Within each channel, exposure times and contrast settings were consistent for each membrane.
Blocking buffers for fluorescence western blotting
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 right signal-to-noise ratio.
| Thermo Fisher Scientific product | Description | Cat. No. | |
|---|---|---|---|
| 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 | 37538 (PBS) 37542 (TBS) |
| Specialty | Fish Serum Blocking Buffer | Fish serum, less likely to have nonspecific binding with antibodies and other mammalian proteins | 37527 |
Stripping buffers for fluorescent western blotting
Reprobing a stripped blot helps save 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 overwhelm 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 | Cat. No. |
|---|---|---|
| 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 |
Fluorescent western blot reagents
Alexa Fluor Plus secondary antibodies
Image capture and analysis for fluorescent western blots
iBright Imaging Systems
Capture and analyze publication quality images from up to 4-plex multiplexed fluorescent western blots with the iBright FL1500 Imaging System. This high-performance instrument enhances the imaging experience through powerful hardware, advanced automated technologies, and an interface that is easy to use for researchers of all experience levels.
Fluorescent western blot optimized reagents
To limit background fluorescence, specific fluorescent western blotting reagents have been developed and optimized to help you obtain excellent 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 can optimize exposure to the bright dye front. We recommended using fluorescence-friendly sample buffers without bromophenol blue.
| Thermo Fisher Scientific product | Description | Cat. No. |
|---|---|---|
| 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 | PageRuler Plus Prestained Protein Ladder | PageRuler Prestained NIR Protein Ladder | |
|---|---|---|---|
| Applications | Fluorescence, chemiluminescent, direct visualization | NIR and RGB fluorescence, direct visualization | NIR fluorescence, direct visualization |
| Molecular weight range | ~10-250 kDa | ~10-250 kDa | ~11-250 kDa |
| Number of bands | 12 | 9 | 10 |
| No. of colors | 10 single colored bands (prestained) | 9 colored bands- six blue, one green and two orange bands (prestained) | 10 single colored bands (prestained) |
| Major features | Two unstained proteins (30 and 80 kDa) with IgG binding sites for chemiluminescent or fluorescent detection | Three colors for quick reference of approximate size | Reference band at 55kDa with greater intensity |
| Fluorescence wavelength | NIR and determined by 2° antibodies used | 700 nm and 550nm | NIR |
| Cat. No. | LC5615 | 26619 | 26635 |
Membranes for fluorescent western blotting
To eliminate a major source of background fluorescence, use transfer membranes with low autofluorescence across all channels. While nitrocellulose membranes (e.g., Cat. No. 88018) typically exhibit lower background fluorescence than PVDF, low fluorescence PVDF membranes (e.g., Cat. No. 67800) perform well in all common fluorescent channels enabling flexible multiplexing strategies.
| Thermo Fisher Scientific product | Pore size | Format |
|---|---|---|
| Low fluorescence PVDF | 0.3 µm | Roll (8.3 cm x 10 m) Pre-cut sheets, mini (8.3 x 7.3 cm) Pre-cut sheets, midi (13.5 x 8.3 cm) Membrane/filter paper sandwiches, mini (8.3 x 7.3 cm) Membrane/filter paper sandwiches, midi (13.5 x 8.3 cm) |
| Nitrocellulose | 0.45 µm | Roll (30 cm x 3.5 m) Pre-cut sheets, mini (8 x 8 cm) Pre-cut sheets, midi (8 x 12 cm) |
| Nitrocellulose | 0.2 µm | Pre-cut sheets, mini (8 x 8 cm) Pre-cut sheets, midi (12 x 8 cm) |
Figure 1.Invitrogen Low Fluorescence PVDF exhibits lower autofluorescence when compared to nitrocellulose and standard PVDF membranes. Unprocessed membranes were hydrated and imaged together in six different fluorescent channels on a compatible digital western blot imaging system. Within each channel, exposure times and contrast settings were consistent for each membrane.
Blocking buffers for fluorescence western blotting
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 right signal-to-noise ratio.
| Thermo Fisher Scientific product | Description | Cat. No. | |
|---|---|---|---|
| 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 | 37538 (PBS) 37542 (TBS) |
| Specialty | Fish Serum Blocking Buffer | Fish serum, less likely to have nonspecific binding with antibodies and other mammalian proteins | 37527 |
Stripping buffers for fluorescent western blotting
Reprobing a stripped blot helps save 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 overwhelm 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 | Cat. No. |
|---|---|---|
| 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 |
Fluorescent western blot reagents
Alexa Fluor Plus secondary antibodies
Image capture and analysis for fluorescent western blots
iBright Imaging Systems
Capture and analyze publication quality images from up to 4-plex multiplexed fluorescent western blots with the iBright FL1500 Imaging System. This high-performance instrument enhances the imaging experience through powerful hardware, advanced automated technologies, and an interface that is easy to use for researchers of all experience levels.
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
For Research Use Only. Not for use in diagnostic procedures.