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Transfer Membrane Improves Fluorescent Detection of Proteins
Background fluorescent emission of Immobilon-FL (blue) to nitrocellulose and PVDF at commonly used excitation wavelengths. |
By Darshan Koticha, Masaharu Mabuchi, and Michael Carelli
The Western blot (or immunoblot) is a widely used analytical tool for detecting specific proteins in a complex mixture. Proteins are separated based on their relative size using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) then transferred to a membrane, usually nitrocellulose or polyvinylidene difluoride (PVDF), where an accurate replica is obtained.1 The transferred proteins may be visualized using total protein stains such as SYPRO Ruby stains or protein-specific immunodetection.
Specific target proteins are typically visualized using an indirect immunoassay technique, where the select protein is targeted by a primary antibody. The bound primary antibody is then visualized by a secondary antibody obtained from a different animal species. To enable visualization, the secondary antibody often is conjugated to an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) for chemiluminescent or chromogenic detection. Alternatively, it can be chemically labeled with a radioisotope for autoradiographic detection or with a fluorophore for fluorescent detection. The use of enzyme-conjugated secondary antibodies provides signal amplification that results in increased sensitivity. Additionally, a single secondary antibody can be used to detect different primary antibodies from the same species. Various detection methods are described in detail below.
Radioactive detection —When the detection antibody is labeled with a
radioisotope, such as 125I or 35S, the emitted radiation can be detected by X-ray
film or phosphor screens. The use of this method has declined due to the development
of alternative technologies and concerns about handling and disposal. Chromogenic
detection — HRP or AP enzymes conjugated to the secondary antibody catalyze the
formation of a colored precipitate from a soluble substrate. Due to its relatively
low sensitivity, this method is useful only for detecting high-abundance proteins.
Chemiluminescent detection — Today’s most common visualization technique
exploits the HRP and AP antibody conjugates used in chromogenic detection but
applies them to chemiluminescent detection. HRP and AP catalyze a reaction that
results in the emission of light. This method’s high sensitivity enables protein
detection down to the low picogram range. Ease of use, flexibility and the incorporation
of non-hazardous chemicals make it popular compared to radioactive techniques.
Fluorescent detection — Recently, there has been a trend towards the
application of fluorescence-based detection of Western blots. This approach provides
the benefits of greater signal stability as well as the capacity for semi-quantitative
and multiplex analyses. In addition, the emergence of highly photostable dyes
and enhanced imaging instrumentation has improved the sensitivity of fluorescent
immunodetection.
Fluorescent molecules (fluorophores) absorb light at one wavelength and emit it at a higher wavelength. Each fluorophore has a characteristic absorption and emission spectrum. Many of them are available for conjugation to antibodies. Fluorescence has been used routinely for the detection of proteins in cells (immunocytochemistry), tissues (immunohistochemistry), and for nucleic acid detection by fluorescent in-situ hybridization (FISH). A major impediment to the widespread adoption of fluorescent detection methodologies for Western blotting has been the lack of a membrane with low background fluorescence.
Immobilon-FL low auto-fluorescence PVDF membrane (Millipore Corp., Billerica, MA) exhibits low background fluorescence and enables high signal-to-noise values. Sensitivities down to the low picogram range have been reported using this membrane in conjunction with quantum dot nanocrystals.2
This 0.45 mm pore size transfer membrane was developed as a “drop-in” to standard Western blotting protocols. The membrane is compatible with all common transfer and blocking buffers as well as total protein stains. Western blots can be imaged using fluorescence ranging from ultraviolet to infrared. Single color detection or multiplexing can be performed efficiently on the membrane.
Western blots of serial dilutions of human serum transferrin using an Alexa 430-conjugated secondary antibody.
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Low-background membrane performance
Figure 1 shows the membrane’s low background compared to conventional PVDF and nitrocellulose at the commonly used excitation/emission wavelengths for fluorophores, such as Coumarin, Fluorescein isothiocyanate (FITC), Rhodamine and Texas Red. Fluorescence was measured using a Tecan SPECTRAFluor Plus fluorometer (TECAN, Durham, NC). The background of standard PVDF and nitrocellulose membranes is up to 40- and 5-fold higher, respectively, than this low fluorescence membrane. The difference is especially significant when membranes were excited with ultraviolet or blue light. These data demonstrate that this membrane may be used for detecting fluorophores in any part of the ultraviolet to infrared spectrum.
Figure 2 illustrates that low fluorescence background results in improved detection limits and sensitivity. In this experiment, serum transferrin was detected on the low fluorescing membrane and other commercially available blotting membranes. Serum was loaded in a two-fold dilution series from left to right. Blots were probed with a goat anti-transferrin primary antibody (Sigma, St. Louis, MO) followed by an Alexa 430 conjugated rabbit anti-goat secondary antibody (Invitrogen, Carlsbad, CA). The membranes were scanned using the Storm 840 imaging system (GE-Amersham, Piscataway, NJ) in the blue fluorescence mode (excitation 450 nm and emission 520 nm). The results show that the limit of detection increased on this membrane, compared to nitrocellulose or standard PVDF membranes. The high background of the other membranes tends to obscure the signal, which makes quantitation of signal intensities more difficult.
Multiplex detection
In order to detect two or more target proteins using traditional detection methods, multiple gels have to be run, or blots have to be stripped and reprobed a number of times. Stripping, which is not recommended for nitrocellulose membranes, may cause a disproportionate loss of protein on the membranes leading to reduced sensitivity and signal loss. Moreover, it is inconvenient for researchers to run multiple blots since they may be limited by sample availability.
Fluorescent detection methods enable the simultaneous detection of two or more proteins on the same blot, known as multiplexing. Multiplexing enables a variety of analyses, including differential protein expression and post-translational modifications such as phosphorylation and glycosylation. It is critical that the primary and secondary antibody pairs are well-characterized with low non-specific binding for signal clarity. Additionally, the fluorescent labels on the antibodies must be separated spectrally from each other so that the signal emitted from one fluorophore does not overlap with the other. The use of the extremely photostable and efficient nanocrystals (Invitrogen, Qdot conjugates) has improved multiplexed detection significantly.(2) Qdot nanocrystals are up to 50 times brighter than conventional fluorophores and extremely photostable. They are excited in the ultra-violet range and exhibit narrow emissions ranging from 525 to 800 nm, with less than 5% overlap between different Qdot nanocrystals.
Multiplexed detection of total (pseudocolored green) and phosphorylated (red) MAPK in extracts of PDGF-treated NIH 3T3 cells, as a function of time (A). Western blots of serial dilutions of E. coli lysates overexpressing GST protein (green) with either a c-myc (purple) or an HA (red). (Provided courtesy of Invitrogen.)
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Figure 3A shows a time course of the phosphorylation of MAP Kinase (MAPK) following platelet-derived-growth-factor (PDGF) treatment of serum-starved NIH 3T3 cells. Total MAPK was detected using a primary mouse anti-pan-MAPK and a secondary goat anti-mouse Qdot 605 nm conjugate (pseudo-colored green, panel 1). Phosphorylated MAPK was detected using a primary rabbit anti-phospho-MAPK and a secondary goat anti-rabbit Qdot 705 nm conjugate (pseudo-colored red, panel 2). Panel 3 is an overlay of panels 1 and 2, which shows the relative amounts of phosphorylated and MAPK proteins in the PDGF-treated 3T3 cells. Thus, fluorescence allows the multiplexed detection of phosphorylated and total protein on the same blot.
Figure 3B is a three-color Western blot of serial dilutions of E. coli lysates over-expressing glutathione-s-transferase (GST) with either a c-myc or an HA tag. The c-myc tag was detected using a mouse primary antibody, followed by a secondary goat anti-mouse Qdot 705 (pseudo-colored purple, panel 1) conjugate. GST was detected using a goat anti-GST antibody directly conjugated to Qdot 565 (green, panel 2) antibody. The HA tag was detected using a rabbit primary antibody, followed by a goat anti-rabbit Qdot 605 (red, panel 3) antibody. Panel 4 is an overlay of the three panels showing the relative amounts of HA and c-myc tagged GST in the total lysate. This blot shows that three different proteins can be simultaneously detected on the same gel.
The data show that multiplexed assays for detecting three or more proteins on the same blot are possible using this membrane. A previous paper also reported a three-color Western blot using secondary antibodies labeled with FITC, Cy3 and Cy5 dyes.3 Since this membrane has lower background than nitrocellulose, especially in the ultraviolet spectrum, a four or even five-color Western blot is possible.
Conclusion
The Immobilon-FL membrane enables researchers to adapt their Western blotting systems to sensitive and photostable, fluorescent chemistries. It retains all the advantages of PVDF membranes, including durability, high protein adsorption and retention, while increasing the signal-to-noise ratio in fluorescent Western blots. The low background of the membrane enables sensitivities of detection in the low picogram range,(2) compared to 6 nanograms on nitrocellulose membranes,(4) representing a 100-fold increase in sensitivity. Thus, sensitivities of detection on the low fluorescing membrane approach chemiluminescent detection methods.
About the Author: Darshan Koticha, Masaharu Mabuchi, and Michael Carelli (corresponding author michael_carelli@millipore.com) are with Millipore Corp. in Billerica, MA.
References
1. Towbin, H., Staehelin, T. and Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 76:4350-4 (1979).
2. Ornberg, R.L., Harper, T.F. and Liu, H. Western blot analysis with quantum dot fluorescent technology: a sensitive and quantitative method for multiplexed proteomics. Nature Methods 2:79-81 (2005).
3. Gingrich, J.C., Davis, D.R. and Nguyen, Q. Multiplex detection and quantitation of proteins on Western blots using fluorescent probes. Biotechniques 29:636-42 (2000).
4. Fradelizi, J., Friederich, E., Beckerle, M.C. and R.M. Golsteyn. Quantitative measurement of proteins by Western blotting with Cy5-coupled secondary antibodies. Biotechniques 26:484-6, 488, 490 passim (1999).
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