4-plex fluorescent western blot imaged on iBright Imaging Systems

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 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 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 productDescriptionCat. No.
Fluorescent Compatible Sample BufferFluorescent 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 ladderPageRuler Plus Prestained protein ladderPageRuler Prestained NIR protein ladder
 iBright Prestained Protein LadderPageRuler Plus Prestained Protein LadderPageRuler Prestained NIR Protein Ladder
ApplicationsFluorescence, chemiluminescent, direct visualizationNIR and RGB fluorescence, direct visualizationNIR fluorescence, direct visualization
Molecular weight range~10-250 kDa~10-250 kDa~11-250 kDa
Number of bands12910
No. of colors10 single colored bands (prestained)9 colored bands- six blue, one green and two orange bands (prestained)10 single colored bands (prestained)
Major featuresTwo unstained proteins (30 and 80 kDa) with IgG binding sites for chemiluminescent or fluorescent detectionThree colors for quick reference of approximate sizeReference band at 55kDa with greater intensity
Fluorescence wavelengthNIR and determined by 2° antibodies used700 nm and 550nmNIR
Cat. No.LC56152661926635

Membranes for fluorescent western blotting

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 productDescriptionOrder
Low-Fluorescence PVDF0.2 µm22860 (7cm x 8.4cm)
Nitrocellulose0.45 µm88018 (30cm x 3.5m)
77010 (8cm x 12cm)
88025 (8cm x 8cm)
88014 (7.9cm x 10.5cm)
Nitrocellulose0.2 µm77012 (8cm x 12cm)
88024 (8cm x 8cm)
88013 (7.9cm x 10.5cm)

See a complete list of all our available membranes

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 best signal-to-noise ratio.

 Thermo Fisher Scientific productDescriptionCat. No.
Start withBlocker FL Blocking BufferDesigned to reduce cross-reactivity and produce high signal-to-noise ratios37565
NextStartingBlock Blocking BufferSingle 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 reagents37538 (PBS)
37542 (TBS)
SpecialtyFish Serum Blocking BufferFish serum, less likely to have nonspecific binding with antibodies and other mammalian proteins37527

Stripping buffers for fluorescent western blotting

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 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 productDescriptionCat. No.
Restore Fluorescent Western Blot Stripping BufferA gentle and highly effective reagent for quickly removing primary and near-infrared (IR) dye-labeled secondary antibodies from western blots transferred on PVDF membranes62300

Fluorescent western blot reagents

Catalog # Name Size Price (USD) Qty
LC2570 Fluorescent Compatible Sample Buffer (4X, non-reducing) Each
84.65

Online exclusive

88.25
Save 3.60 (4%)
84788 Pierce™ LDS Sample Buffer, Non-Reducing (4X) Each
51.50
LC5615 iBright™ Prestained Protein Ladder, 2 x 250 μL Each
313.65

Online exclusive

327.00
Save 13.35 (4%)
LC5602 MagicMark™ XP Western Protein Standard, 250 μL Each
377.00
22860 Low-Fluorescence PVDF Transfer Membranes, 0.2 µm, 7 x 8.4 cm Each
140.65

Online exclusive

160.00
Save 19.35 (12%)
88018 Nitrocellulose Membranes, 0.45 μm, 1 Roll Each
510.00
77012 Nitrocellulose Membranes, 0.2 μm, 25 Pre-cut Membranes Each
350.65

Online exclusive

394.00
Save 43.35 (11%)
37565 Blocker™ FL Fluorescent Blocking Buffer (10X) Each
147.65

Online exclusive

161.00
Save 13.35 (8%)
37538 StartingBlock™ (PBS) Blocking Buffer, 1 L Each
232.65

Online exclusive

262.00
Save 29.35 (11%)
37542 StartingBlock™ (TBS) Blocking Buffer, 1 L Each
235.65

Online exclusive

263.00
Save 27.35 (10%)
37527 Fish Serum Blocking Buffer, 500 mL Each
229.65

Online exclusive

257.00
Save 27.35 (11%)
26619 PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa, 2 x 250 μL Each
162.65

Online exclusive

192.00
Save 29.35 (15%)
62300 Restore™ Fluorescent Western Blot Stripping Buffer, 100 mL Each
183.65

Online exclusive

199.00
Save 15.35 (8%)

Alexa Fluor Plus secondary antibodies

Catalog # Name Size Price (USD) Qty
A32723 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 1 mg
298.50

Special offer

Online exclusive

Ends: 30-Mar-2025

398.00
Save 99.50 (25%)
A32727 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 555 1 mg
303.00

Special offer

Online exclusive

Ends: 30-Mar-2025

404.00
Save 101.00 (25%)
A32728 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 647 1 mg
298.50

Special offer

Online exclusive

Ends: 30-Mar-2025

398.00
Save 99.50 (25%)
A32729 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 680 1 mg
298.50

Special offer

Online exclusive

Ends: 30-Mar-2025

398.00
Save 99.50 (25%)
A32730 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 800 1 mg
298.50

Special offer

Online exclusive

Ends: 30-Mar-2025

398.00
Save 99.50 (25%)
A32731 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 1 mg
294.75

Special offer

Online exclusive

Ends: 30-Mar-2025

393.00
Save 98.25 (25%)
A32732 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 555 1 mg
298.50

Special offer

Online exclusive

Ends: 30-Mar-2025

398.00
Save 99.50 (25%)
A32733 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 647 1 mg
298.50

Special offer

Online exclusive

Ends: 30-Mar-2025

398.00
Save 99.50 (25%)
A32734 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 680 1 mg
298.50

Special offer

Online exclusive

Ends: 30-Mar-2025

398.00
Save 99.50 (25%)
A32735 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 800 1 mg
300.00

Special offer

Online exclusive

Ends: 30-Mar-2025

400.00
Save 100.00 (25%)

Image capture and analysis for fluorescent western blots

iBright FL1500 Imaging system for fluorescent western blot documentation

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.

Explore our western blot documentation systems

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 productDescriptionCat. No.
Fluorescent Compatible Sample BufferFluorescent 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 ladderPageRuler Plus Prestained protein ladderPageRuler Prestained NIR protein ladder
 iBright Prestained Protein LadderPageRuler Plus Prestained Protein LadderPageRuler Prestained NIR Protein Ladder
ApplicationsFluorescence, chemiluminescent, direct visualizationNIR and RGB fluorescence, direct visualizationNIR fluorescence, direct visualization
Molecular weight range~10-250 kDa~10-250 kDa~11-250 kDa
Number of bands12910
No. of colors10 single colored bands (prestained)9 colored bands- six blue, one green and two orange bands (prestained)10 single colored bands (prestained)
Major featuresTwo unstained proteins (30 and 80 kDa) with IgG binding sites for chemiluminescent or fluorescent detectionThree colors for quick reference of approximate sizeReference band at 55kDa with greater intensity
Fluorescence wavelengthNIR and determined by 2° antibodies used700 nm and 550nmNIR
Cat. No.LC56152661926635

Membranes for fluorescent western blotting

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 productDescriptionOrder
Low-Fluorescence PVDF0.2 µm22860 (7cm x 8.4cm)
Nitrocellulose0.45 µm88018 (30cm x 3.5m)
77010 (8cm x 12cm)
88025 (8cm x 8cm)
88014 (7.9cm x 10.5cm)
Nitrocellulose0.2 µm77012 (8cm x 12cm)
88024 (8cm x 8cm)
88013 (7.9cm x 10.5cm)

See a complete list of all our available membranes

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 best signal-to-noise ratio.

 Thermo Fisher Scientific productDescriptionCat. No.
Start withBlocker FL Blocking BufferDesigned to reduce cross-reactivity and produce high signal-to-noise ratios37565
NextStartingBlock Blocking BufferSingle 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 reagents37538 (PBS)
37542 (TBS)
SpecialtyFish Serum Blocking BufferFish serum, less likely to have nonspecific binding with antibodies and other mammalian proteins37527

Stripping buffers for fluorescent western blotting

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 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 productDescriptionCat. No.
Restore Fluorescent Western Blot Stripping BufferA gentle and highly effective reagent for quickly removing primary and near-infrared (IR) dye-labeled secondary antibodies from western blots transferred on PVDF membranes62300

Fluorescent western blot reagents

Catalog # Name Size Price (USD) Qty
LC2570 Fluorescent Compatible Sample Buffer (4X, non-reducing) Each
84.65

Online exclusive

88.25
Save 3.60 (4%)
84788 Pierce™ LDS Sample Buffer, Non-Reducing (4X) Each
51.50
LC5615 iBright™ Prestained Protein Ladder, 2 x 250 μL Each
313.65

Online exclusive

327.00
Save 13.35 (4%)
LC5602 MagicMark™ XP Western Protein Standard, 250 μL Each
377.00
22860 Low-Fluorescence PVDF Transfer Membranes, 0.2 µm, 7 x 8.4 cm Each
140.65

Online exclusive

160.00
Save 19.35 (12%)
88018 Nitrocellulose Membranes, 0.45 μm, 1 Roll Each
510.00
77012 Nitrocellulose Membranes, 0.2 μm, 25 Pre-cut Membranes Each
350.65

Online exclusive

394.00
Save 43.35 (11%)
37565 Blocker™ FL Fluorescent Blocking Buffer (10X) Each
147.65

Online exclusive

161.00
Save 13.35 (8%)
37538 StartingBlock™ (PBS) Blocking Buffer, 1 L Each
232.65

Online exclusive

262.00
Save 29.35 (11%)
37542 StartingBlock™ (TBS) Blocking Buffer, 1 L Each
235.65

Online exclusive

263.00
Save 27.35 (10%)
37527 Fish Serum Blocking Buffer, 500 mL Each
229.65

Online exclusive

257.00
Save 27.35 (11%)
26619 PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa, 2 x 250 μL Each
162.65

Online exclusive

192.00
Save 29.35 (15%)
62300 Restore™ Fluorescent Western Blot Stripping Buffer, 100 mL Each
183.65

Online exclusive

199.00
Save 15.35 (8%)

Alexa Fluor Plus secondary antibodies

Catalog # Name Size Price (USD) Qty
A32723 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 1 mg
298.50

Special offer

Online exclusive

Ends: 30-Mar-2025

398.00
Save 99.50 (25%)
A32727 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 555 1 mg
303.00

Special offer

Online exclusive

Ends: 30-Mar-2025

404.00
Save 101.00 (25%)
A32728 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 647 1 mg
298.50

Special offer

Online exclusive

Ends: 30-Mar-2025

398.00
Save 99.50 (25%)
A32729 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 680 1 mg
298.50

Special offer

Online exclusive

Ends: 30-Mar-2025

398.00
Save 99.50 (25%)
A32730 Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 800 1 mg
298.50

Special offer

Online exclusive

Ends: 30-Mar-2025

398.00
Save 99.50 (25%)
A32731 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 1 mg
294.75

Special offer

Online exclusive

Ends: 30-Mar-2025

393.00
Save 98.25 (25%)
A32732 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 555 1 mg
298.50

Special offer

Online exclusive

Ends: 30-Mar-2025

398.00
Save 99.50 (25%)
A32733 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 647 1 mg
298.50

Special offer

Online exclusive

Ends: 30-Mar-2025

398.00
Save 99.50 (25%)
A32734 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 680 1 mg
298.50

Special offer

Online exclusive

Ends: 30-Mar-2025

398.00
Save 99.50 (25%)
A32735 Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 800 1 mg
300.00

Special offer

Online exclusive

Ends: 30-Mar-2025

400.00
Save 100.00 (25%)

Image capture and analysis for fluorescent western blots

iBright FL1500 Imaging system for fluorescent western blot documentation

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.

Explore our western blot documentation systems

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.