荧光 Western Blot 检测 | Thermo Fisher Scientific

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
Molecular weight markers
Membranes
Blocking buffers
Stripping buffers

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	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.

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.

Suggested fluorescent friendly blocking buffers

Western reagents