5 Steps to Multiplexed Fluorescent Western Blotting

Multiplex fluorescent western blotting provides accurate, quantitative results, stable signals, and the ability to clearly evaluate multiple protein targets on a single blot, which makes this technique increasingly popular. With a range of fluorescent dyes and antibodies for western blot detection, as well as the availability of new imaging systems, multiplexed western blotting can save time, reduce cost, and improve efficiency of data generation and collection.

Here, we divide multiplexed fluorescent western blotting into 5 steps from sample preparation to data collection to help you optimize each step, and avoid pitfalls, in order to achieve your best results.

Follow these 5 Steps to Multiplexed Fluorescent Western Blots

Sample preparation for protein gel electrophoresis

Protein samples used for western blotting can be obtained from many sources, including tissues, cells, and subcellular fractions. Many methods are available for isolating and purifying proteins from these sources.

It is important when preparing protein samples for fluorescent western blotting experiments to avoid to introduction of contaminants that will contribute to background fluorescence. Bromophenol blue will contribute to background fluorescence during imaging.

Total protein extraction reagents and kits provide extracts that are compatible with western blotting are shown below:

Sample type Goal Recommended Thermo Scientific reagents or kits
Primary cultured or mammalian cells or tissues Total protein extraction

M-PER reagent
T-PER reagent
N-PER reagent
RIPA Lysis and Extraction Buffer
Pierce IP Lysis Buffer
Cultured mammalian cells or tissues Subcellular fractionation or organelle isolation

NE-PER reagent
Subcellular Fractionation Kits
Mitochondria Isolation Kits
Pierce Cell Surface Protein Isolation Kit
Syn-PER reagent
Lysosomes Enrichment Kit
Bacterial cells Total protein extraction B-PER reagent
Yeast cells Total protein extraction

Y-PER reagent
Y-PER Plus reagent
Insect cells (baculovirus) Total protein extraction

I-PER reagent
Plant tissue (leaf, stem, root, flower) Total protein extraction

P-PER reagent

Find related products


Protein quantitation

Knowing the concentration of your protein sample will help you determine the proper amount to load on a gel during the electrophoresis step. A wide variety of protein assays is available for measuring the protein concentration of your sample. Use the Protein Assay Selection Guide to help you determine the right protein assay for your needs.


In addition to reliable protein quantification and analysis, perform UV-Vis photometric research applications such as DNA and RNA analysis and ELISAs with the Thermo Scientific Multiskan Sky Microplate Spectrophotometer. The Multiskan Sky reader features a broad wavelength range (200-1000 nm), path length correction and a fast reading speed. The optional µDrop plate enables microvolume analysis—it's like performing up to 16 NanoDrop measurements at once! The intuitive touchscreen user interface, on-board software and built-in protocols let you run quick measurements directly from the instrument. Alternatively, with any instrument purchase you can use our unlimited license, easy-to use Thermo Scientific SkanIt Software with access to our extensive online library of ready-made protocols.

Multiskan Sky Microplate Spectrophotometer with µDrop plate

Resources

Separate proteins based on molecular weight using gel electrophoresis

Separation of prepared protein samples using protein gel electrophoresis is the second step of multiplexed fluorescent western blotting. Obtaining good separation of the target proteins from one another can be achieved by careful selection of protein gel chemistry and polyacrylamide concentration. Consider using gradient gels, which provide a polyacrylamide concentration gradient that helps provide sharper banding patterns.


Choose the right protein gel

Obtaining optimal resolution of your target protein can be achieved by choosing the right of protein gel chemistry for your protein. Our precast protein gels are offered in four different chemistries. The choice of whether to use one chemistry or another depends on the abundance of the protein you’re separating, the size of the protein.

Find the right gel for your protein
Convert from other suppliers' gels

Find the right protein gel and buffers for optimal resolution of your protein.

Protein sample type Gel chemistry Precast gel Sample buffer* Protein ladders Running buffer Transfer buffer
Low-abundance/ posttranslationally modified

Broad-range MW (6–400 kDa)
Bis-Tris Bolt Bis-Tris Plus (load up to 60 μL) Fluorescent Compatible Sample Buffer iBright Prestained Protein Ladder
Bolt MOPS SDS Buffer (15–260 kDa); Bolt MES SDS Buffer (3.5–160 kDa); add Bolt Antioxidant for reduced samples Bolt Transfer Buffer
NuPAGE Bis-Tris NuPAGE MOPS SDS Buffer (15–260 kDa); Bolt MES SDS Buffer (3.5–160 kDa); add Bolt Antioxidant for reduced samples NuPAGE Transfer Buffer
High-abundance

Broad-range MW (6–400 kDa)
Tris-glycine Novex Tris-Glycine WedgeWell format Novex Tris-Glycine SDS Running Buffer Novex Tris-Glycine Transfer Buffer
High MW (40–500 kDa) Tris-acetate NuPAGE Tris-Acetate Gels Spectra Multicolor High Range Protein Ladder NuPAGE Tris-Acetate SDS Running Buffer NuPAGE Transfer Buffer
Low MW (2.5–40 kDa) Tricine Novex Tricine Mini Gels Spectra Multicolor Low Range Protein Ladder Novex Tricing SDS Running Buffer Novex Tris-Glycine Transfer Buffer
*Heat samples at 70°C for 10 min.

Sample Buffers


Protein ladders

iBright Prestained Protein Ladder is recommended for use as a molecular weight marker for multiplexed fluorescent western blotting of medium–range molecular weight proteins. It allows direct visualization of protein bands during electrophoresis and features:

iBright Prestained Protein Ladder as it appears in visible light and with chemiluminescent and fluorescent detection methods
  • Ten blue-stained proteins, also fluorophore labeled to enable direct and near-IR fluorescent visualization
  • Two unstained proteins (30 kDa and 80 kDa) that enable direct visualization of chemiluminescent or fluorescent detection

Standard prestained molecular weight markers can be used, but the loading amount will need to be optimized if the marker contains fluorescent bands since overloading can increase background fluorescence and signal bleed-through to adjacent lanes. iBright Prestained Protein Ladder allows you to decrease the amount of molecular weight markers loaded onto the gel: typically, 2–4 μL is sufficient for visualization and fluorescence detection.

Learn more about the iBright Prestained Protein Ladder


Protein gels welcome packs

iBright Prestained Protein Ladder as it appears in visible light and with chemiluminescent and fluorescent detection methods

Protein gels welcome packs are a cost effective way to start using Invitrogen precast protein gels for multiplexed fluorescent western blotting experiments. Protein gels welcome packs are available for each of the protein gel chemistries and come with precast protein mini gels, buffers, ladders, and a Mini Gel Tank to provide you with great savings over the cost of the individual components. iBright Prestained Protein Ladder and Invitrogen Fluorescent Compatible Sample Buffer (Cat. No. LC2570) are not included with protein gels welcome packs and must be ordered separately.

Learn more about the protein gels welcome packs

Resources

Transfer the protein from the gel matrix to a solid-support membrane

The efficient transfer of proteins from the polyacrylamide gel after electrophoresis to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane is an important step in western blotting so that specific proteins can be detected using immunodetection techniques.

Electrophoretic methods of gel transfer can be sorted into wet transfer, semi-dry transfer, and dry transfer.

Use the selection table below to compare transfer methods and needed equipment, buffers, and time requirements.

  Traditional wet transfer Semi-dry transfer Dry transfer
 

Mini Blot Module



Power Blotter



Power Blotter XL

iBlot 2 Gel Transfer Device
Transfer time 60 min 5–10 min 5–10 min 7 min
Capacity of device 1 mini gel (per module) or 2 mini gels (two modules per tank) 2 mini gels or
1 midi gel
4 mini gels or
2 midi gels
2 mini gels or
1 midi gel
Blotting area 9 x 9 cm 10 x 18 cm 21 x 22.5 cm 8.5 x 13.5 cm
Requires transfer buffer Yes, (200–400 mL per module) Depends on consumable choice:
Yes – if using Pre-Cut Membranes and Filters (50 mL per mini gel or 100 mL per midi gel)
No – if using ready-to-use Select Transfer Stacks
Depends on consumable choice:
Yes – if using Pre-Cut Membranes and Filters (50 mL per mini gel or 100 mL per midi gel)
No – if using ready-to-use Select Transfer Stacks
No
Power supply External Internal Internal Internal
  Order Now Order Now Order Now Order Now

Video: See how easy it is to do a 7-minute protein gel transfer with the iBlot 2 Gel Transfer Device.

Transfer membranes

To eliminate a major source of background fluorescence, use membranes with low autofluorescence, including nitrocellulose and specialty low-fluorescence PVDF membranes such as Thermo Scientific Nitrocellulose Membrane (Cat. No. 88018) and Low-Fluorescence PVDF Transfer Membrane (Cat. No. 22860). Only handle membranes with gloved hands and clean blunt forceps to limit contamination and scratches on the membranes, which can contribute to background fluorescence and artifacts.

Tip: Avoid using pens on membranes, as many inks fluoresce. Use a pencil instead.


Transfer buffers

Use the table below to identify the correct transfer buffer for your protein gel.

Protein sample type Gel chemistry Precast gel Transfer buffer
Low-abundance/ posttranslationally modified

Broad-range MW (6–400 kDa)
Bis-Tris Bolt Bis-Tris Plus (load up to 60 μL) Bolt Transfer Buffer
NuPAGE Bis-Tris NuPAGE Transfer Buffer
High-abundance

Broad-range MW (6–400 kDa)
Tris-glycine Novex Tris-Glycine WedgeWell format Novex Tris-Glycine Transfer Buffer
High MW (40–500 kDa) Tris-acetate NuPAGE Tris-Acetate Gels NuPAGE Transfer Buffer
Low MW (2.5–40 kDa) Tricine Novex Tricine Mini Gels Novex Tris-Glycine Transfer Buffer

Resources

Detect transferred proteins with fluorescently labelled antibodies

Careful selection of antibodies and fluorescent labels is required for successful multiplexed fluorescent western blotting. Download our application note: Fluorescent western blotting - a guide to multiplexing for a full discussion of antibody and fluorescent label selection.

Choosing fluorescent labels

Select fluorophores with optically distinct spectra to avoid cross-channel fluorescence. Examples of fluorophores chosen for multiplex experiments for distinct excitation spectra and emission spectra are shown in figures 1 and 2, below. See Table 1 for examples of multiplex fluorophore combinations that can be used with the iBright FL1000 Imaging System for 1- to 4-probe multiplexed western experiments.

Table 1. Examples of fluorophore combinations for successful multiplexing with the iBright FL Imaging System.

Number of targets Conjugate 1 Conjugate 2 Conjugate 3 Conjugate 4
1 Alexa Fluor Plus 647      
2 Alexa Fluor Plus 647 Alexa Fluor 546    
3 Alexa Fluor Plus 647 Alexa Fluor 546
Alexa Fluor Plus 488  
4 Alexa Fluor Plus 647 Alexa Fluor 546
Alexa Fluor Plus 488 Alexa Fluor Plus 800
Example of multiplex experiment with carefully chosen fluorophores with distinct excitation spectra

Figure 1. Example of multiplex experiment with carefully chosen fluorophores with distinct excitation spectra. In this example generated on the Fluorescence SpectraViewer, the excitation spectra (dashed lines) of Alexa Fluor Plus 488 and Alexa Fluor 546 fluorophores have minimal overlap within the range of the excitation filter. Despite both fluorophores having part of their emission spectra (solid lines) within the range of the emission filter, Alexa Fluor 546 would not be excited by the excitation filter that has been selected for Alexa Fluor Plus 488, so no fluorescence from Alexa Fluor 546 would be present to go through the emission filter.

Example of multiplex experiment with carefully chosen fluorophores with distinct emission spectra

Figure 2. Example of multiplex experiment with carefully chosen fluorophores with distinct emission spectra. In this example generated on the Fluorescence SpectraViewer, the emission spectra (solid lines) of Alexa Fluor Plus 680 and Alexa Fluor 790 have no overlap within the ranges of the two emission filters. Despite both fluorophores having part of their excitation spectra (dashed lines) within the range of excitation filter 1, any Alexa Fluor 790 fluorescence generated by that excitation range is not within the wavelengths allowed to pass through emission filter 1, so no fluorescence from Alexa Fluor 790 would reach the camera detector in that channel.

Alexa Fluor Plus Secondary Antibodies

Alexa Fluor Plus Secondary Antibodies

Need to detect and visualize low-abundance targets in rare or precious samples by fluorescent western blot?

Compare to traditional Alexa Fluor secondary antibodies:

  • Higher signal-to-noise ratio
  • Lower cross-reactivity

Learn more

Search for specific antibodies

Find antibodies of interest using the search tool below. Then filter the results by target or host species, monoclonal or polyclonal antibody type, application, and other criteria.

Search now


Find related products


Automation of the detection process

ibind

There is a hands-free alternative for all blocking, antibody incubation, and washing steps using our Invitrogen iBind and iBind Flex Western Devices. Find out more at thermofisher.com/ibind.


Troubleshooting

Problem Possible cause Solution
Weak or no signal Insufficient amount of primary antibody
  • Increase primary antibody concentration
  • Ensure primary antibody has a good titer and is specific for the antigen to be detected
  • For a low-abundance target in a cell or tissue lysate, increase the amount of primary antibody or the amount of sample loaded on the gel
  • Extend the incubation time to overnight at 4°C, or 3–6 hours at room temperature
  • Try using an antibody enhancer
Lost activity of antibody
  • Ensure the antibody was stored appropriately
  • Check the expiration date of the antibody
  • Avoid multiple uses of pre-diluted antibodies
Exposure time is too short
  • Increase exposure time
  • Utilize the Smart Exposure freature to obtain an optimal image on the iBright FL1000 system
Incorrect instrument settings
  • Ensure the correct excitation and emission ranges are selected for the intended fluorophore
Use of detergent
  • Too much detergent or the nature of the detergent can result in washing away the signal—decrease or eliminate detergent
Blocking buffer blocks antigen
  • Some blocking solutions can mask the blot and reduce the availability of the antigen to the antibody, especially if the blocking step is >1 hour
  • Dilute your primary antibody in wash buffer
  • Evaluate another blocking buffer
Quantity of sample loaded on the gel
  • Too much lysate can overcrowd your specific target and reduce the antibody sensitivity
  • Too little lysate leads to insufficient availability of the target of interest
  • Perform serial dilutions of the lysate or sample to determine the optimal amount of protein to load
Poor transfer of protein or loss of the protein after transfer
  • Check transfer conditions to confirm protein transfer
  • Reoptimization may be required when probing for a new protein
Nonspecific bands Poor antibody specificity for the target of interest
  • Evaluate additional primary antibodies
Poor sample integrity
  • Sample degradation due to overheating or protease activity results in target breakdown and low target recognition by the antibody
Antibody cross-reactivity in multiplex detection
  • Carefully choose secondary antibody host, and avoid pairing a mouse and a rat secondary antibody
  • Use highly cross-adsorbed secondary antibody conjugates
  • Reduce the amount of the secondary antibody used to remain within the optimal performance range
Fluorescent bleed-through from another channel when multiplexing (appearance of an unexpected band)
  • Avoid spectrally close conjugates, especially when the signal is very strong
  • Ensure that your fluorescent dyes can be distinctively detected on your imager
  • Use the autoexposure feature on the instrument to determine the optimal exposure time for each channel
Background issues (high, uneven, or speckled) High background due to membrane contamination
  • Handle the membrane carefully using clean dishes or trays and clean forceps
  • Determine the best blocking buffer for your application—primary antibodies will react differently in different blocking buffers; blocking buffers like normal animal sera or milk may result in cross-reactivity
Artifacts from overloading the protein marker or ladder
  • Decrease the molecular weight marker loaded on the gel
Nonoptimal wash or diluent solutions
  • Use a wash buffer with 0.1–0.2% Tween 20
  • Prepare the secondary antibody diluent with 0.05% Tween 20
  • Increase the number of wash steps or the time per step; insufficient washing can result in background signal
High background from an excess of secondary antibody
  • Optimize the secondary antibody dilution depending on the dye being used following the vendorrecommended dilution and adapting accordingly
Blotchy or uneven background due to the membrane drying out
  • Ensure good coverage of the whole blot during all incubation steps
  • Ensure consistent agitation during every incubation step
Incorrect choice of membrane
  • The nature of the membrane can affect the background; for example, PVDF membranes can generate autofluorescence and cause high background, so use low-fluorescence PVDF membranes
Speckles and fingerprints on the membrane
  • Use clean forceps to handle the membrane and avoid directly touching the membrane; particulates and contaminants from unclean tools will fluoresce and disturb the detection of the signal of interest
  • Use clean incubation trays or dishes—rinsing with methanol followed by water will help dissolve residual dried dyes from previous uses
  • Clean transfer devices and dusty consumables (e.g., pads) if using a wet transfer method as they can introduce speckles
  • Clean the imager surface with ethanol to remove dust, lint, and residues before capturing the image

Resources

Imaging of the fluorescent western blot and quantitation of proteins

With the latest advances in imaging software and instrument sensitivity, fluorescent image capture and quantitative western blot analysis is now easier. Be certain the instrument you plan to use is capable of capturing the number of fluors you would like to detect and whether the imager has the correct filters for use with the fluors.

We offer iBright FL1000 Imaging System, a powerful, easy-to-use system, which provides sensitive, streamlined, multimode image capture (see image 2 below). The iBright FL1000 is capable of easily capturing 4-plex images. It features a large capacitive touch-screen interface and intelligently designed software.

See product details for the iBright FL1000 Imaging System
Download the iBright FL1000 Imaging System brochure


Four-channel imaging of a multiplexed fluorescent western blot
Figure 2. Four-channel imaging of a multiplexed fluorescent western blot. Up to four different proteins can be imaged simultaneously on the same blot. HA-tagged RB-1 was expressed in HeLa cell extract using the 1-Step Human High-Yield Mini IVT Kit (Cat. No. 88890) and appropriate expression-ready clones. The resulting reaction mixture was prepared for reducing SDS-PAGE, serially diluted, and electrophoresed on a Novex WedgeWell 4–20% Tris-glycine gel (Cat. No. XP04200PK2). The protein was transferred to a PVDF membrane using the Pierce Power Blotter (Cat. No. 22834), and the membrane was blocked and probed with the following primary antibodies: chicken anti-calreticulin (Cat. No. PA1-903), rabbit anti-HSP90 (Cat. No. PA3-013), and mouse anti-p23 (Cat. No. MA3-414). The membrane was washed and probed with the following secondary antibodies in TBS-Tween 20: goat anti-chicken Alexa Fluor 546 (Cat. No. A11040) (pseudocolored in yellow), goat anti-rabbit Alexa Fluor Plus 800 (Cat. No. A32735) (pseudocolored in green), and goat anti-mouse Alexa Fluor Plus 680 (Cat. No. A32729) (pseudocolored in red). The membrane was again washed and probed for 1 hr with mouse anti-HA primary antibody directly conjugated to Alexa Fluor 488 (Cat. No. 26183-D488) (pseudocolored in blue), in TBS-Tween 20. The membrane was washed and imaged on the iBright FL1000 Imaging System.

Quantitation

Normalization is a critical step in obtaining reliable and reproducible quantitative western blotting. Under ideal conditions, normalization would not be necessary, but factors such as sample loading and transfer efficiency make normalizing the western blot essential. Download this technical note, which provides the basic principles of normalization using internal loading controls and describes how to accurately normalize western blots to obtain meaningful, reproducible data.

Application note: Normalization in western blotting to achieve relative quantitation


Reprobing fluorescent western blots

To reprobe the blot with other antibodies, use Restore Fluorescent Western Blot Stripping Buffer. Restore Fluorescent Western Blot Stripping Buffer enables the reuse of PVDF membranes, simplifying the Western blot optimization process and allowing the same blot to be reprobed with different primary antibodies to detect alternative targets. Restore Fluorescent Western Blot Stripping Buffer is for use with low-fluorescence PVDF membrane only.

See product details for Restore Fluorescent Western Blot Stripping Buffer

iWestern Workflow

Need a start-to-finish western blotting solution?
Or just looking to boost the performance of one of the main blotting steps?

Check out the iWestern workflow

Sample preparation for protein gel electrophoresis

Protein samples used for western blotting can be obtained from many sources, including tissues, cells, and subcellular fractions. Many methods are available for isolating and purifying proteins from these sources.

It is important when preparing protein samples for fluorescent western blotting experiments to avoid to introduction of contaminants that will contribute to background fluorescence. Bromophenol blue will contribute to background fluorescence during imaging.

Total protein extraction reagents and kits provide extracts that are compatible with western blotting are shown below:

Sample type Goal Recommended Thermo Scientific reagents or kits
Primary cultured or mammalian cells or tissues Total protein extraction

M-PER reagent
T-PER reagent
N-PER reagent
RIPA Lysis and Extraction Buffer
Pierce IP Lysis Buffer
Cultured mammalian cells or tissues Subcellular fractionation or organelle isolation

NE-PER reagent
Subcellular Fractionation Kits
Mitochondria Isolation Kits
Pierce Cell Surface Protein Isolation Kit
Syn-PER reagent
Lysosomes Enrichment Kit
Bacterial cells Total protein extraction B-PER reagent
Yeast cells Total protein extraction

Y-PER reagent
Y-PER Plus reagent
Insect cells (baculovirus) Total protein extraction

I-PER reagent
Plant tissue (leaf, stem, root, flower) Total protein extraction

P-PER reagent

Find related products


Protein quantitation

Knowing the concentration of your protein sample will help you determine the proper amount to load on a gel during the electrophoresis step. A wide variety of protein assays is available for measuring the protein concentration of your sample. Use the Protein Assay Selection Guide to help you determine the right protein assay for your needs.


In addition to reliable protein quantification and analysis, perform UV-Vis photometric research applications such as DNA and RNA analysis and ELISAs with the Thermo Scientific Multiskan Sky Microplate Spectrophotometer. The Multiskan Sky reader features a broad wavelength range (200-1000 nm), path length correction and a fast reading speed. The optional µDrop plate enables microvolume analysis—it's like performing up to 16 NanoDrop measurements at once! The intuitive touchscreen user interface, on-board software and built-in protocols let you run quick measurements directly from the instrument. Alternatively, with any instrument purchase you can use our unlimited license, easy-to use Thermo Scientific SkanIt Software with access to our extensive online library of ready-made protocols.

Multiskan Sky Microplate Spectrophotometer with µDrop plate

Resources

Separate proteins based on molecular weight using gel electrophoresis

Separation of prepared protein samples using protein gel electrophoresis is the second step of multiplexed fluorescent western blotting. Obtaining good separation of the target proteins from one another can be achieved by careful selection of protein gel chemistry and polyacrylamide concentration. Consider using gradient gels, which provide a polyacrylamide concentration gradient that helps provide sharper banding patterns.


Choose the right protein gel

Obtaining optimal resolution of your target protein can be achieved by choosing the right of protein gel chemistry for your protein. Our precast protein gels are offered in four different chemistries. The choice of whether to use one chemistry or another depends on the abundance of the protein you’re separating, the size of the protein.

Find the right gel for your protein
Convert from other suppliers' gels

Find the right protein gel and buffers for optimal resolution of your protein.

Protein sample type Gel chemistry Precast gel Sample buffer* Protein ladders Running buffer Transfer buffer
Low-abundance/ posttranslationally modified

Broad-range MW (6–400 kDa)
Bis-Tris Bolt Bis-Tris Plus (load up to 60 μL) Fluorescent Compatible Sample Buffer iBright Prestained Protein Ladder
Bolt MOPS SDS Buffer (15–260 kDa); Bolt MES SDS Buffer (3.5–160 kDa); add Bolt Antioxidant for reduced samples Bolt Transfer Buffer
NuPAGE Bis-Tris NuPAGE MOPS SDS Buffer (15–260 kDa); Bolt MES SDS Buffer (3.5–160 kDa); add Bolt Antioxidant for reduced samples NuPAGE Transfer Buffer
High-abundance

Broad-range MW (6–400 kDa)
Tris-glycine Novex Tris-Glycine WedgeWell format Novex Tris-Glycine SDS Running Buffer Novex Tris-Glycine Transfer Buffer
High MW (40–500 kDa) Tris-acetate NuPAGE Tris-Acetate Gels Spectra Multicolor High Range Protein Ladder NuPAGE Tris-Acetate SDS Running Buffer NuPAGE Transfer Buffer
Low MW (2.5–40 kDa) Tricine Novex Tricine Mini Gels Spectra Multicolor Low Range Protein Ladder Novex Tricing SDS Running Buffer Novex Tris-Glycine Transfer Buffer
*Heat samples at 70°C for 10 min.

Sample Buffers


Protein ladders

iBright Prestained Protein Ladder is recommended for use as a molecular weight marker for multiplexed fluorescent western blotting of medium–range molecular weight proteins. It allows direct visualization of protein bands during electrophoresis and features:

iBright Prestained Protein Ladder as it appears in visible light and with chemiluminescent and fluorescent detection methods
  • Ten blue-stained proteins, also fluorophore labeled to enable direct and near-IR fluorescent visualization
  • Two unstained proteins (30 kDa and 80 kDa) that enable direct visualization of chemiluminescent or fluorescent detection

Standard prestained molecular weight markers can be used, but the loading amount will need to be optimized if the marker contains fluorescent bands since overloading can increase background fluorescence and signal bleed-through to adjacent lanes. iBright Prestained Protein Ladder allows you to decrease the amount of molecular weight markers loaded onto the gel: typically, 2–4 μL is sufficient for visualization and fluorescence detection.

Learn more about the iBright Prestained Protein Ladder


Protein gels welcome packs

iBright Prestained Protein Ladder as it appears in visible light and with chemiluminescent and fluorescent detection methods

Protein gels welcome packs are a cost effective way to start using Invitrogen precast protein gels for multiplexed fluorescent western blotting experiments. Protein gels welcome packs are available for each of the protein gel chemistries and come with precast protein mini gels, buffers, ladders, and a Mini Gel Tank to provide you with great savings over the cost of the individual components. iBright Prestained Protein Ladder and Invitrogen Fluorescent Compatible Sample Buffer (Cat. No. LC2570) are not included with protein gels welcome packs and must be ordered separately.

Learn more about the protein gels welcome packs

Resources

Transfer the protein from the gel matrix to a solid-support membrane

The efficient transfer of proteins from the polyacrylamide gel after electrophoresis to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane is an important step in western blotting so that specific proteins can be detected using immunodetection techniques.

Electrophoretic methods of gel transfer can be sorted into wet transfer, semi-dry transfer, and dry transfer.

Use the selection table below to compare transfer methods and needed equipment, buffers, and time requirements.

  Traditional wet transfer Semi-dry transfer Dry transfer
 

Mini Blot Module



Power Blotter



Power Blotter XL

iBlot 2 Gel Transfer Device
Transfer time 60 min 5–10 min 5–10 min 7 min
Capacity of device 1 mini gel (per module) or 2 mini gels (two modules per tank) 2 mini gels or
1 midi gel
4 mini gels or
2 midi gels
2 mini gels or
1 midi gel
Blotting area 9 x 9 cm 10 x 18 cm 21 x 22.5 cm 8.5 x 13.5 cm
Requires transfer buffer Yes, (200–400 mL per module) Depends on consumable choice:
Yes – if using Pre-Cut Membranes and Filters (50 mL per mini gel or 100 mL per midi gel)
No – if using ready-to-use Select Transfer Stacks
Depends on consumable choice:
Yes – if using Pre-Cut Membranes and Filters (50 mL per mini gel or 100 mL per midi gel)
No – if using ready-to-use Select Transfer Stacks
No
Power supply External Internal Internal Internal
  Order Now Order Now Order Now Order Now

Video: See how easy it is to do a 7-minute protein gel transfer with the iBlot 2 Gel Transfer Device.

Transfer membranes

To eliminate a major source of background fluorescence, use membranes with low autofluorescence, including nitrocellulose and specialty low-fluorescence PVDF membranes such as Thermo Scientific Nitrocellulose Membrane (Cat. No. 88018) and Low-Fluorescence PVDF Transfer Membrane (Cat. No. 22860). Only handle membranes with gloved hands and clean blunt forceps to limit contamination and scratches on the membranes, which can contribute to background fluorescence and artifacts.

Tip: Avoid using pens on membranes, as many inks fluoresce. Use a pencil instead.


Transfer buffers

Use the table below to identify the correct transfer buffer for your protein gel.

Protein sample type Gel chemistry Precast gel Transfer buffer
Low-abundance/ posttranslationally modified

Broad-range MW (6–400 kDa)
Bis-Tris Bolt Bis-Tris Plus (load up to 60 μL) Bolt Transfer Buffer
NuPAGE Bis-Tris NuPAGE Transfer Buffer
High-abundance

Broad-range MW (6–400 kDa)
Tris-glycine Novex Tris-Glycine WedgeWell format Novex Tris-Glycine Transfer Buffer
High MW (40–500 kDa) Tris-acetate NuPAGE Tris-Acetate Gels NuPAGE Transfer Buffer
Low MW (2.5–40 kDa) Tricine Novex Tricine Mini Gels Novex Tris-Glycine Transfer Buffer

Resources

Detect transferred proteins with fluorescently labelled antibodies

Careful selection of antibodies and fluorescent labels is required for successful multiplexed fluorescent western blotting. Download our application note: Fluorescent western blotting - a guide to multiplexing for a full discussion of antibody and fluorescent label selection.

Choosing fluorescent labels

Select fluorophores with optically distinct spectra to avoid cross-channel fluorescence. Examples of fluorophores chosen for multiplex experiments for distinct excitation spectra and emission spectra are shown in figures 1 and 2, below. See Table 1 for examples of multiplex fluorophore combinations that can be used with the iBright FL1000 Imaging System for 1- to 4-probe multiplexed western experiments.

Table 1. Examples of fluorophore combinations for successful multiplexing with the iBright FL Imaging System.

Number of targets Conjugate 1 Conjugate 2 Conjugate 3 Conjugate 4
1 Alexa Fluor Plus 647      
2 Alexa Fluor Plus 647 Alexa Fluor 546    
3 Alexa Fluor Plus 647 Alexa Fluor 546
Alexa Fluor Plus 488  
4 Alexa Fluor Plus 647 Alexa Fluor 546
Alexa Fluor Plus 488 Alexa Fluor Plus 800
Example of multiplex experiment with carefully chosen fluorophores with distinct excitation spectra

Figure 1. Example of multiplex experiment with carefully chosen fluorophores with distinct excitation spectra. In this example generated on the Fluorescence SpectraViewer, the excitation spectra (dashed lines) of Alexa Fluor Plus 488 and Alexa Fluor 546 fluorophores have minimal overlap within the range of the excitation filter. Despite both fluorophores having part of their emission spectra (solid lines) within the range of the emission filter, Alexa Fluor 546 would not be excited by the excitation filter that has been selected for Alexa Fluor Plus 488, so no fluorescence from Alexa Fluor 546 would be present to go through the emission filter.

Example of multiplex experiment with carefully chosen fluorophores with distinct emission spectra

Figure 2. Example of multiplex experiment with carefully chosen fluorophores with distinct emission spectra. In this example generated on the Fluorescence SpectraViewer, the emission spectra (solid lines) of Alexa Fluor Plus 680 and Alexa Fluor 790 have no overlap within the ranges of the two emission filters. Despite both fluorophores having part of their excitation spectra (dashed lines) within the range of excitation filter 1, any Alexa Fluor 790 fluorescence generated by that excitation range is not within the wavelengths allowed to pass through emission filter 1, so no fluorescence from Alexa Fluor 790 would reach the camera detector in that channel.

Alexa Fluor Plus Secondary Antibodies

Alexa Fluor Plus Secondary Antibodies

Need to detect and visualize low-abundance targets in rare or precious samples by fluorescent western blot?

Compare to traditional Alexa Fluor secondary antibodies:

  • Higher signal-to-noise ratio
  • Lower cross-reactivity

Learn more

Search for specific antibodies

Find antibodies of interest using the search tool below. Then filter the results by target or host species, monoclonal or polyclonal antibody type, application, and other criteria.

Search now


Find related products


Automation of the detection process

ibind

There is a hands-free alternative for all blocking, antibody incubation, and washing steps using our Invitrogen iBind and iBind Flex Western Devices. Find out more at thermofisher.com/ibind.


Troubleshooting

Problem Possible cause Solution
Weak or no signal Insufficient amount of primary antibody
  • Increase primary antibody concentration
  • Ensure primary antibody has a good titer and is specific for the antigen to be detected
  • For a low-abundance target in a cell or tissue lysate, increase the amount of primary antibody or the amount of sample loaded on the gel
  • Extend the incubation time to overnight at 4°C, or 3–6 hours at room temperature
  • Try using an antibody enhancer
Lost activity of antibody
  • Ensure the antibody was stored appropriately
  • Check the expiration date of the antibody
  • Avoid multiple uses of pre-diluted antibodies
Exposure time is too short
  • Increase exposure time
  • Utilize the Smart Exposure freature to obtain an optimal image on the iBright FL1000 system
Incorrect instrument settings
  • Ensure the correct excitation and emission ranges are selected for the intended fluorophore
Use of detergent
  • Too much detergent or the nature of the detergent can result in washing away the signal—decrease or eliminate detergent
Blocking buffer blocks antigen
  • Some blocking solutions can mask the blot and reduce the availability of the antigen to the antibody, especially if the blocking step is >1 hour
  • Dilute your primary antibody in wash buffer
  • Evaluate another blocking buffer
Quantity of sample loaded on the gel
  • Too much lysate can overcrowd your specific target and reduce the antibody sensitivity
  • Too little lysate leads to insufficient availability of the target of interest
  • Perform serial dilutions of the lysate or sample to determine the optimal amount of protein to load
Poor transfer of protein or loss of the protein after transfer
  • Check transfer conditions to confirm protein transfer
  • Reoptimization may be required when probing for a new protein
Nonspecific bands Poor antibody specificity for the target of interest
  • Evaluate additional primary antibodies
Poor sample integrity
  • Sample degradation due to overheating or protease activity results in target breakdown and low target recognition by the antibody
Antibody cross-reactivity in multiplex detection
  • Carefully choose secondary antibody host, and avoid pairing a mouse and a rat secondary antibody
  • Use highly cross-adsorbed secondary antibody conjugates
  • Reduce the amount of the secondary antibody used to remain within the optimal performance range
Fluorescent bleed-through from another channel when multiplexing (appearance of an unexpected band)
  • Avoid spectrally close conjugates, especially when the signal is very strong
  • Ensure that your fluorescent dyes can be distinctively detected on your imager
  • Use the autoexposure feature on the instrument to determine the optimal exposure time for each channel
Background issues (high, uneven, or speckled) High background due to membrane contamination
  • Handle the membrane carefully using clean dishes or trays and clean forceps
  • Determine the best blocking buffer for your application—primary antibodies will react differently in different blocking buffers; blocking buffers like normal animal sera or milk may result in cross-reactivity
Artifacts from overloading the protein marker or ladder
  • Decrease the molecular weight marker loaded on the gel
Nonoptimal wash or diluent solutions
  • Use a wash buffer with 0.1–0.2% Tween 20
  • Prepare the secondary antibody diluent with 0.05% Tween 20
  • Increase the number of wash steps or the time per step; insufficient washing can result in background signal
High background from an excess of secondary antibody
  • Optimize the secondary antibody dilution depending on the dye being used following the vendorrecommended dilution and adapting accordingly
Blotchy or uneven background due to the membrane drying out
  • Ensure good coverage of the whole blot during all incubation steps
  • Ensure consistent agitation during every incubation step
Incorrect choice of membrane
  • The nature of the membrane can affect the background; for example, PVDF membranes can generate autofluorescence and cause high background, so use low-fluorescence PVDF membranes
Speckles and fingerprints on the membrane
  • Use clean forceps to handle the membrane and avoid directly touching the membrane; particulates and contaminants from unclean tools will fluoresce and disturb the detection of the signal of interest
  • Use clean incubation trays or dishes—rinsing with methanol followed by water will help dissolve residual dried dyes from previous uses
  • Clean transfer devices and dusty consumables (e.g., pads) if using a wet transfer method as they can introduce speckles
  • Clean the imager surface with ethanol to remove dust, lint, and residues before capturing the image

Resources

Imaging of the fluorescent western blot and quantitation of proteins

With the latest advances in imaging software and instrument sensitivity, fluorescent image capture and quantitative western blot analysis is now easier. Be certain the instrument you plan to use is capable of capturing the number of fluors you would like to detect and whether the imager has the correct filters for use with the fluors.

We offer iBright FL1000 Imaging System, a powerful, easy-to-use system, which provides sensitive, streamlined, multimode image capture (see image 2 below). The iBright FL1000 is capable of easily capturing 4-plex images. It features a large capacitive touch-screen interface and intelligently designed software.

See product details for the iBright FL1000 Imaging System
Download the iBright FL1000 Imaging System brochure


Four-channel imaging of a multiplexed fluorescent western blot
Figure 2. Four-channel imaging of a multiplexed fluorescent western blot. Up to four different proteins can be imaged simultaneously on the same blot. HA-tagged RB-1 was expressed in HeLa cell extract using the 1-Step Human High-Yield Mini IVT Kit (Cat. No. 88890) and appropriate expression-ready clones. The resulting reaction mixture was prepared for reducing SDS-PAGE, serially diluted, and electrophoresed on a Novex WedgeWell 4–20% Tris-glycine gel (Cat. No. XP04200PK2). The protein was transferred to a PVDF membrane using the Pierce Power Blotter (Cat. No. 22834), and the membrane was blocked and probed with the following primary antibodies: chicken anti-calreticulin (Cat. No. PA1-903), rabbit anti-HSP90 (Cat. No. PA3-013), and mouse anti-p23 (Cat. No. MA3-414). The membrane was washed and probed with the following secondary antibodies in TBS-Tween 20: goat anti-chicken Alexa Fluor 546 (Cat. No. A11040) (pseudocolored in yellow), goat anti-rabbit Alexa Fluor Plus 800 (Cat. No. A32735) (pseudocolored in green), and goat anti-mouse Alexa Fluor Plus 680 (Cat. No. A32729) (pseudocolored in red). The membrane was again washed and probed for 1 hr with mouse anti-HA primary antibody directly conjugated to Alexa Fluor 488 (Cat. No. 26183-D488) (pseudocolored in blue), in TBS-Tween 20. The membrane was washed and imaged on the iBright FL1000 Imaging System.

Quantitation

Normalization is a critical step in obtaining reliable and reproducible quantitative western blotting. Under ideal conditions, normalization would not be necessary, but factors such as sample loading and transfer efficiency make normalizing the western blot essential. Download this technical note, which provides the basic principles of normalization using internal loading controls and describes how to accurately normalize western blots to obtain meaningful, reproducible data.

Application note: Normalization in western blotting to achieve relative quantitation


Reprobing fluorescent western blots

To reprobe the blot with other antibodies, use Restore Fluorescent Western Blot Stripping Buffer. Restore Fluorescent Western Blot Stripping Buffer enables the reuse of PVDF membranes, simplifying the Western blot optimization process and allowing the same blot to be reprobed with different primary antibodies to detect alternative targets. Restore Fluorescent Western Blot Stripping Buffer is for use with low-fluorescence PVDF membrane only.

See product details for Restore Fluorescent Western Blot Stripping Buffer

iWestern Workflow

Need a start-to-finish western blotting solution?
Or just looking to boost the performance of one of the main blotting steps?

Check out the iWestern workflow