ReadiView™ Biotin: All-In-One Biotinylation & Quantification of Biotin Labeling
Fundamentals of Flow Cytometry
ReadiUse™ Lyophilized Phycobiliproteins
Buccutite™ Conjugation Technology
AssayWise Letters Vol. 8(1)
Phycobiliproteins are a family of photosynthetic light-harvesting proteins derived from microalgae and cyanobacteria. These proteins have covalently attached linear tetrapyrrole groups, known as phycobilins, which play a critical role in capturing light energy. In microalgae and cyanobacteria, energy absorbed by these phycobilins is efficiently transferred via fluorescence resonance energy transfer (FRET), to chlorophyll pigments for use in photosynthetic reactions.
Compared to chemically synthetic fluorescent dyes, phycobiliproteins emit intense fluorescence signals due to their relatively high fluorescence quantum yields and extinction coefficients. Because their fluorescence is not quenched by biomolecules, phycobiliproteins can serve as valuable fluorescence tags in numerous applications. Phycobiliproteins-conjugated to biological molecules (e.g. immunoglobulin, protein A or streptavidin) have found great success in flow cytometry, fluorescence activated cell sorting (FACS), histochemistry, imaging and to a limited degree reactive oxygen species detection. Two main classes of phycobiliproteins are commercially available phycoerythrin (PE) and allophycocyanin (APC).
PE (Cat# 2558) exhibits an intensely bright yellow-orange fluorescence with an extinction coefficient (ε) of 1,960,000 cm-1M-1 and a quantum yield (Φ) of 0.84. In comparison to Cy3® (Cat# 141), which has an extinction coefficient (ε) of 150,000 cm-1M-1 and a quantum yield (Φ) of 0.24, PE is significantly brighter. Its absorbance spectrum, which is characterized by three absorption bands, shows a primary absorbance peak at 565 nm with two secondary peaks at 496 nm and 545 nm. The relative prominence of the secondary peaks varies significantly among PE’s derived from different species.
Phycoerythrin is available in two forms. R-phycoerythrin (R-PE), which is isolated from RhodophytaI, and B-phycoerythrin (B-PE), which is isolated from Bangiales. Both B-PE and R-PE are comprised of three types of subunits: α (~20,000 daltons), β (~20,000 daltons) and γ (~30,000 daltons). While they share similar subunit structure, the chromophore content of their subunits differs causing differences in the relative intensities of their absorbance peaks.
Figure 1. Comparison of absorbance and emission spectra of R-PE and B-PE.
Table 1. Spectral Properties of R-PE and B-PE.
|Absorption maximum||565 nm||545nm|
|Additional Absorption peak||498 nm||563nm|
|Emission maximum||573 nm||572nm|
|Extinction Coefficient (ε)||1.96 x 106 M-1cm-1||2.41 x 106 M-1cm-1|
|Quantum Yield (QY)||0.84||0.98|
|Brightness (ε x QY)||1.65 x 106 M-1cm-1||2.36 x 106 M-1cm-1|
|Specifications||A566/A280 ≥ 5.0||A545/A280 ≥ 5.5|
|A566/A498 < 1.5||A620/A546 ≤ 0.01|
|A620/A566 < 0.01|
APC (Cat# 2554) exhibits a bright far-red fluorescence with an extinction coefficient of 700,000 cm-1M-1 and a quantum yield (Φ) of 0.68. In comparison to Cy5® (Cat# 151), which has an extinction coefficient (ε = 250,000 cm-1M-1) and quantum yield (Φ = 0.20) of Cy5®, APC is significantly burgher. It has as a primary absorbance maximum at 652 nm with a secondary maximum at 625 nm. APC has a fluorescence emission maximum at 662 nm, which is in the red region of the visible spectrum.
Figure 2. Excitation and emission spectra of APC.
Table 2. Spectral Properties of APC.
|Extinction Coefficient (ε)||7.3 x 105 M-1cm-1|
|Quantum Yield (QY)||0.68|
|Brightness (ε x QY)||4.9 x 105 M-1cm-1|
|Specifications||A650/A620 ≥ 1.25|
|A650/A280 ≥ 4.5|
|Cross-link Ratio ≥ 1.0|
ReadiUse™ PE & APC
In order to use PE or APC for flow cytometry, the fluorescenct protein must first be conjugated to an antibody. However, commercially available phycobiliproteins come concentrated in an ammonium sulfate suspension. Although both exhibit good long-term stability, they require tedious purification processes, such as dialysis, sulfate precipitation or gel filtration chromatography, to remove the ammonium sulfate impurities prior to antibody conjugation. Once purified PE and APC must be stored refrigerated, not frozen, and consumed within one month for optimal results.
AAT Bioquest has developed an innovative technique for the lyophilization of purified phycobiliproteins. Our line of ReadiUse™ phycobiliproteins are supplied as lyophilized powders free of ammonium sulfate. Removal of this strong precipitating agent eliminates purification processes, and facilitates the rapid conjugation of PE or APC to antibodies and other proteins. The following is an overview of a protocol for ReadiUse™ PE (Cat# 2500) using Buccutite™ Rapid Protein Crosslinking Kit (Cat# 1315):
- Reconstitute ReadiUse™ PE in H2O
- Activate PE and antibody with Buccutite™ conjugation linkers
- Mix Buccutite activated PE and activated antibody
- Purify Conjugates using desalting column
Comparative Analysis: ReadiUse™ vs The Competition
In the present study, lyophilized ReadiUse™ PE (Cat# 2500) and ReadiUse™ APC (Cat# 2503) (AAT Bioquest) were stored under various conditions and subsequently tested for performance against traditional phycobiliproteins, suspended in ammonium sulfate. Prior to testing, traditional phycobiliproteins were purified to remove ammonium sulfate. Comparisons were made for spectral property, fluorescence quantum yields and labeling efficiencies.
Storage Conditions Tested
ReadiUse™ PE and ReadiUse™ APC were aliquoted into 1 mg vials and stored at four different temperatures for duration of two months. Temperatures include:
- Fresh – Freshly purified phycobiliproteins suspended in ammonium sulfate (not stored, prepared on day of experiemnt)
- L – 4oC – Lyophilized powder, ReadiUse™ PE and ReadiUse™ APC stored at 2 – 8oC
- L – RT – Lyophilized powder, ReadiUse™ PE and ReadiUse™ APC stored at room temperature
- L – (-20oC) – Lyophilized powder, ReadiUse™ PE and ReadiUse™ APC stored at < - 15 oC
- L – (-65oC) – Lyophilized powder, ReadiUse™ PE and ReadiUse™ APC stored a < -65oC
Preparing PE & APC
On test day, phycobiliproteins saturated in ammonium sulfate solution were purified using dialysis to remove ammonium sulfate impurities. ReadiUse™ PE and ReadiUse™ APC were removed from storage and reconstituted with 100 μL H2O for a final concentration of 10 mg/mL.
ReadiUse™ PE Spectral Properties
The absorbance and emission spectra of ReadiUse™ PE and freshly purified PE in PBS, were measured under identical conditions and concentrations. The spectra comparison data is summarized in Table 3, Table 4 and Figure 3. Data illustrates that ReadiUse™ PE satisfies all the specifications of PE, is very stable and can be stored from RT to -65oC for ~2 months with little variation in excitation, emission and quantum yields.
Table 3. Spectral properties comparision of ReadiUse™ PE and ammonium-sulfate supplied PE.
|PE||Fresh||L-4oC||L-RT||L- (-20oC)||L- (-65oC)||Specification|
Table 4. Quantum yield of ReadiUse™ PE and ammonium-sulfate supplied PE.
|Relative Quantum Yield||100%||99.5%||97.8%||100%||99.3%|
Figure 3. Comparison of lyophilized (ReadiUse™) PE and liquid form PE. Absorbance ratios of 565 to 280nm and 565 to 498 nm were compared and the relative emission intensity at the same concentration were also measured with 490 nm excitation and plotted in the figure.
ReadiUse™ APC Spectral Properties
The absorbance and emission spectra of ReadiUse™ APC and freshly purified APC in PBS, were measured under identical conditions and concentrations. The spectra comparison data is summarized in Table 5, Table 6 and Figure 4. Data illustrates that ReadiUse™ APC satisfies all the specifications of APC, is very stable and can be stored from RT to -65oC for ~2 months with little variation in excitation, emission and quantum yields.
Table 5. Spectral properties comparision of ReadiUse™ APC and ammonium-sulfate supplied APC.
|EM Max||661.00 nm||660.00 nm||660.00 nm||660.00 nm||660.00 nm|
Table 6. Quantum yield of ReadiUse™ APC and ammonium-sulfate supplied APC.
|Ex WL||630 nm||630 nm||630 nm||630 nm||630 nm|
|Em WL||661.01 nm||660.00 nm||660.00 nm||660.00 nm||660.00 nm|
|Relative Quantum Yield||100%||98.7%||97%||97%||97%|
Figure 4. Comparison of lyophilized (ReadiUse™) APC and liquid form APC. Spectra properties were compared and relative emission intensity at the same concentration were measured with 630 nm excitation.
Labeling Efficiency of Lyophilized ReadiUse™ PE and Liquid Form PE
The labeling efficiency of ReadiUse™ PE is another important factor to examine as it directly affects the conjugate’s fluorescence yield. For example, a common application of PE is to modify it with Cy5 to produce a PE/Cy5 tandem dye. Over-labeling of Cy5 onto PE will significantly reduce fluorescence yield due to anomalously strong quenching.
Labeling efficiency can be determined by calculating the degree of labeling, which is the molar ratio of dye to protein (PE) after conjugation. The reactivity of ReadiUse™ PE was validated using Cy5 conjugation reaction. ReadiUse™ PE, stored at different temperatures (4oC, RT, -20oC and -65oC for 2 months), were reconstituted with 100 μL of H2O for a final concentration of 10 mg/mL. A Cy5, SE (Cat# 151) dye working solution was prepared and added to ReadiUse™ PE solution. The conjugation reaction was done at room temperature for 60 minutes with stirring. Freshly prepared PE was conjugated to Cy5 in the same manner and used as a positive control Figure 5.
Figure 5. Comparison of lyophilized ReadiUse™ PE and Fresh PE labeling efficiencies.
Labeling Efficiency of Lyophilized ReadiUse™ APC and Liquid Form APC
The labeling efficiency of ReadiUse™ APC was validated using Cy7 conjugation reaction. ReadiUse™ APC, stored at different temperatures (4oC, RT, -20oC and -65oC for 2 months), were reconstituted with 100 μL of H2O for a final concentration of 10 mg/mL. A Cy7, SE (Cat# 170) dye working solution was prepared and added to ReadiUse™ APC solution. The conjugation reaction was done at room temperature for 60 minutes with stirring. Freshly prepared APC was conjugated to Cy7 in the same manner and used as a positive control Figure 6.
Figure 6. Comparison of lyophilized ReadiUse™ APC and Fresh APC labeling efficiencies.
The performance of ReadiUse PE-Cy5 tandems and ReadiUse™ APC-Cy7 tandems were tested in HL-60 cells. ReadiUse™ PE-Cy5 tandems and ReadiUse™ APC-Cy7 tandems were conjugated to GXM IgG using Buccutite™ Rapid Protein Conjugation kit (Cat# 1315). Conjugates were also prepared with freshly purified PE-Cy5 and PE-Cy7 tandems using Buccutite™ conjugation kit, and used as a positive control. The results indicated that ReadiUse™ PE-Cy5 and ReadiUse APC-Cy7 conjugates were equivalent in performance to freshly purified PE-Cy5 and APC-Cy7 conjugates.
Figure 7. Comparison the performance of GXM IgG-PE/Cy5 conjugates with ReadiUse™ PE-Cy5 tandems and freshly purified PE-Cy5 tandems. HL-60 cells were stained with or without w6/32 antibody (1ug/ml) for 30 min, and followed by GXM IgG-PE/Cy5 (10ug/ml) for 30min. GXM IgG-PE/Cy5 conjugates were prepared through the same Buccutite™ reaction.
Figure 8. Performance of GXM IgG-APC/Cy7 conjugate prepared with ReadiUse™ APC-Cy7 tandems and freshly purified APC-Cy7 tandems. HL-60 cells were stained with or without w6/32 antibody (1ug/ml) for 30min, and followed by GXM IgG-APC/Cy7 (5ug/ml) for 30min. GXM IgG-APC/Cy7 conjugates were prepared through the same Buccutite™ reaction.
AAT Bioquest’s ReadiUse™ PE and ReadiUse™ APC are on par with commercially available products. Our lyophilized phycobiliproteins are stable at different conditions, such as RT, 4oC and -20oC and -65oC without changes in spectra properties, labeling efficiency and performance. In addition, ReadiUse™ PE and ReadiUse™ APC ammonia sulfate-free powders, do not require any purification (e.g. dialysis or desalting), significantly reduces assay time, increases the consistency in quality and the flexibility in reconstitution concentration, and also decreases the shipping cost.
Product Ordering Information
Table 1. Product ordering information for ReadiUse™ PE, ReadiUse™ APC and ReadiUse™ Tandem dyes.