Search Thermo Fisher Scientific
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.
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.
Sample Buffers
Sample buffers containing bromophenol blue will fluoresce and can contribute to increased background. If using sample buffers with bromophenol blue, the dye front may be run off the gel prior to transfer or cut from the membrane after transfer to avoid background fluorescence signal.
Consider using fluorescence-compatible sample buffers without bromophenol blue, such as Invitrogen Fluorescent Compatible Sample Buffer (Cat. No. LC2570).
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:
- 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.
Protein gels welcome packs
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 or midi gels, buffers, ladders, and a Mini Gel Tank or SureLock Tandem Midi 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.
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.
| Wet transfer | Semi-dry transfer | Dry transfer | |||
|---|---|---|---|---|---|
 |  XCell II Blot Module |  SureLock Tandem Midi Blot Module |  Power Blotter Systems |  iBlot 3 Western Blot Transfer System | |
| Capacity | 1 mini gel per blot module; 1–2 blot modules per tank | Up to 2 mini blots | 1 midi gel per blot module; 1–2 blot modules per tank | 1–4 mini or 1–2 midi gels | 1–2 midi or 1–4 mini gel |
| Transfer time | 60 min | 60–120 min | 30 min | 7–10 min | as few as 3 min |
| Blotting area (cm) | 9 x 9 | 9 x 9 | 9.2 x 14.4 | 10 x 18 or 21 x 22.5 | 8.5 x 13.5 |
| Transfer buffer volume | 220 mL per blot module | 200mL | 300 mL per blot module | Pre-cut membranes & filters: 50–100 mL; Select transfer stacks: buffer not required | Buffer not required |
| Power supply | External | External | External | Internal | Internal |
| Required equipment | Mini Gel Tank: capacity for up to 2 Mini Blot Modules | XCell SureLock Mini-Cell | SureLock Tandem Midi Gel Tank | — | — |
| Learn more | Learn more | Learn more | Learn more | Learn more | |
Video: See how easy it is to do a protein gel transfer with the iBlot 3 Western Blot Transfer System.
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 |
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 FL1500 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 |
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.
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
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
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.
Find related products
Automation of the detection process
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 |
|
| Lost activity of antibody |
| |
| Imaging exposure time is too short |
| |
| Incorrect instrument settings |
| |
| Use of detergent |
| |
| Blocking buffer blocks antigen |
| |
| Insufficient quantity of sample loaded on the gel |
| |
| Poor transfer of protein or loss of the protein after transfer |
| |
| Nonspecific bands | Poor antibody specificity for the target of interest |
|
| Poor sample integrity |
| |
| Antibody cross-reactivity in multiplex detection |
| |
| Fluorescent bleed-through from another channel when multiplexing (appearance of an unexpected band) |
| |
| Background issues (high, uneven, or speckled) | High background due to membrane contamination |
|
| Artifacts from overloading the protein marker or ladder |
| |
| Nonoptimal wash or diluent solutions |
| |
| High background from an excess of secondary antibody |
| |
| Blotchy or uneven background due to the membrane drying out |
| |
| Incorrect choice of membrane |
| |
| Dust and fingerprints on the membrane |
|
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 fluorophores you would like to detect and whether the imager has the correct filters for use with the fluorophores.
We offer iBright FL1500 Imaging System, a powerful, easy-to-use system, which provides sensitive, streamlined, multimode image capture (see image 2 below). The iBright FL1500 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 FL1500 Imaging System
Download the iBright FL1500 Imaging System brochure
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
Protein normalization is a critical step in obtaining reliable and reproducible quantitative western blotting and can be performed using housekeeping proteins or total protein normalization. Under ideal conditions, normalization would not be necessary, but factors such as sample loading and transfer efficiency make normalizing the western blot essential. Total protein normalization is a more reliable method, as housekeeping proteins can be affected by experimental conditions. For total protein normalization of western blotting data, the Invitrogen No-Stain Protein Labeling Reagent is a fast, easy-to-use reagent applied to a membrane after transfer and provides sensitive, linear detection.
The iBright FL1500 Imaging System includes software for effortless normalization using the No-Stain Protein Labeling Reagent and many other methods.
Learn more about the No-Stain Protein Labeling Reagent
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
Need a start-to-finish western blotting solution?
Or just looking to boost the performance of one of the main blotting steps?
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.
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.
Sample Buffers
Sample buffers containing bromophenol blue will fluoresce and can contribute to increased background. If using sample buffers with bromophenol blue, the dye front may be run off the gel prior to transfer or cut from the membrane after transfer to avoid background fluorescence signal.
Consider using fluorescence-compatible sample buffers without bromophenol blue, such as Invitrogen Fluorescent Compatible Sample Buffer (Cat. No. LC2570).
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:
- 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.
Protein gels welcome packs
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 or midi gels, buffers, ladders, and a Mini Gel Tank or SureLock Tandem Midi 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.
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.
| Wet transfer | Semi-dry transfer | Dry transfer | |||
|---|---|---|---|---|---|
| XCell II Blot Module | SureLock Tandem Midi Blot Module | Power Blotter Systems | iBlot 3 Western Blot Transfer System | |
| Capacity | 1 mini gel per blot module; 1–2 blot modules per tank | Up to 2 mini blots | 1 midi gel per blot module; 1–2 blot modules per tank | 1–4 mini or 1–2 midi gels | 1–2 midi or 1–4 mini gel |
| Transfer time | 60 min | 60–120 min | 30 min | 7–10 min | as few as 3 min |
| Blotting area (cm) | 9 x 9 | 9 x 9 | 9.2 x 14.4 | 10 x 18 or 21 x 22.5 | 8.5 x 13.5 |
| Transfer buffer volume | 220 mL per blot module | 200mL | 300 mL per blot module | Pre-cut membranes & filters: 50–100 mL; Select transfer stacks: buffer not required | Buffer not required |
| Power supply | External | External | External | Internal | Internal |
| Required equipment | Mini Gel Tank: capacity for up to 2 Mini Blot Modules | XCell SureLock Mini-Cell | SureLock Tandem Midi Gel Tank | — | — |
| Learn more | Learn more | Learn more | Learn more | Learn more | |
Video: See how easy it is to do a protein gel transfer with the iBlot 3 Western Blot Transfer System.
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 |
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 FL1500 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 |
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.
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
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
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.
Find related products
Automation of the detection process
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 |
|
| Lost activity of antibody |
| |
| Imaging exposure time is too short |
| |
| Incorrect instrument settings |
| |
| Use of detergent |
| |
| Blocking buffer blocks antigen |
| |
| Insufficient quantity of sample loaded on the gel |
| |
| Poor transfer of protein or loss of the protein after transfer |
| |
| Nonspecific bands | Poor antibody specificity for the target of interest |
|
| Poor sample integrity |
| |
| Antibody cross-reactivity in multiplex detection |
| |
| Fluorescent bleed-through from another channel when multiplexing (appearance of an unexpected band) |
| |
| Background issues (high, uneven, or speckled) | High background due to membrane contamination |
|
| Artifacts from overloading the protein marker or ladder |
| |
| Nonoptimal wash or diluent solutions |
| |
| High background from an excess of secondary antibody |
| |
| Blotchy or uneven background due to the membrane drying out |
| |
| Incorrect choice of membrane |
| |
| Dust and fingerprints on the membrane |
|
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 fluorophores you would like to detect and whether the imager has the correct filters for use with the fluorophores.
We offer iBright FL1500 Imaging System, a powerful, easy-to-use system, which provides sensitive, streamlined, multimode image capture (see image 2 below). The iBright FL1500 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 FL1500 Imaging System
Download the iBright FL1500 Imaging System brochure
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
Protein normalization is a critical step in obtaining reliable and reproducible quantitative western blotting and can be performed using housekeeping proteins or total protein normalization. Under ideal conditions, normalization would not be necessary, but factors such as sample loading and transfer efficiency make normalizing the western blot essential. Total protein normalization is a more reliable method, as housekeeping proteins can be affected by experimental conditions. For total protein normalization of western blotting data, the Invitrogen No-Stain Protein Labeling Reagent is a fast, easy-to-use reagent applied to a membrane after transfer and provides sensitive, linear detection.
The iBright FL1500 Imaging System includes software for effortless normalization using the No-Stain Protein Labeling Reagent and many other methods.
Learn more about the No-Stain Protein Labeling Reagent
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
Need a start-to-finish western blotting solution?
Or just looking to boost the performance of one of the main blotting steps?
For Research Use Only. Not for use in diagnostic procedures.