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mFluor™ Blue 590 SE
mFluor™ Blue 590 dye can be well excited with blue laser at 488 nm. It has a huge Stokes shift with emission ~590 nm. mFluor™ Blue 590 dyes are water-soluble, and the protein conjugates prepared with mFluor™ Blue 590 dyes are well excited at 488 nm to give red fluorescence. mFluor™ Blue 590 dye and conjugates are excellent blue laser reagents for flow cytometry detections. Compared to RPE, mFluor™ Blue 590 dyes are much more photostable, making them readily available for fluorescence imaging applications while it is very difficult to use RPE conjugates for fluorescence imaging applications due to the rapid photobleaching of RPE conjugates. It is also a unique fluorochrome for spectral flow cytometry since there are very few existing dyes that have this spectral profile.
Example protocol
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
Note The pH of the protein solution (Solution A) should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer.
Note The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.
Note Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results.
Note The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.
Note Prepare the dye stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.
1. Protein stock solution (Solution A)
Mix 100 µL of a reaction buffer (e.g., 1 M sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) with 900 µL of the target protein solution (e.g. antibody, protein concentration >2 mg/mL if possible) to give 1 mL protein labeling stock solution.Note The pH of the protein solution (Solution A) should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer.
Note The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.
Note Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results.
Note The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.
2. mFluor™ Blue 590 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of mFluor™ Blue 590 SE to make a 10 mM stock solution. Mix well by pipetting or vortex.Note Prepare the dye stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.
SAMPLE EXPERIMENTAL PROTOCOL
This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with mFluor™ Blue 590 SE. You might need further optimization for your particular proteins.
Note Each protein requires distinct dye/protein ratio, which also depends on the properties of dyes. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low dye/protein ratio gives reduced sensitivity.
Note Each protein requires distinct dye/protein ratio, which also depends on the properties of dyes. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low dye/protein ratio gives reduced sensitivity.
Run conjugation reaction
- Use 10:1 molar ratio of Solution B (dye)/Solution A (protein) as the starting point: Add 5 µL of the dye stock solution (Solution B, assuming the dye stock solution is 10 mM) into the vial of the protein solution (95 µL of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD.
Note We recommend to use 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too less or too high, determine the optimal dye/protein ratio at 5:1, 15:1 and 20:1 respectively. - Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes.
Purify the conjugation
The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.- Prepare Sephadex G-25 column according to the manufacture instruction.
- Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
- Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
- Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate.
Note For immediate use, the dye-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses.
Note For longer term storage, dye-protein conjugate solution need be concentrated or freeze dried.
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Correction Factor (260 nm) | Correction Factor (280 nm) |
| mFluor™ Blue 570 SE | 553 | 565 | 1200001 | 0.228 | 0.179 |
| mFluor™ Blue 630 SE | 470 | 632 | 490001 | 0.197 | 0.275 |
| mFluor™ Blue 660 SE | 481 | 663 | 260001 | 0.338 | 0.32 |
| mFluor™ Blue 580 SE | 485 | 580 | 400001 | 0.363 | 0.247 |
| mFluor™ Blue 620 SE | 589 | 616 | 980001 | 0.683 | 0.849 |
| mFluor™ Violet 590 SE | 564 | 591 | 900001 | 0.632 | 0.329 |
| mFluor™ Blue 585 SE | 491 | 578 | 450001 | - | - |
| mFluor™ Blue 583 SE | 498 | 585 | 450001 | 1.17 | 0.35 |
| mFluor™ Blue 615 SE | 510 | 615 | 400001 | - | - |
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References
View all 50 references: Citation Explorer
Multi-set Pre-processing of Multicolor Flow Cytometry Data.
Authors: Folcarelli, Rita and Tinnevelt, Gerjen H and Hilvering, Bart and Wouters, Kristiaan and van Staveren, Selma and Postma, Geert J and Vrisekoop, Nienke and Buydens, Lutgarde M C and Koenderman, Leo and Jansen, Jeroen J
Journal: Scientific reports (2020): 9716
Authors: Folcarelli, Rita and Tinnevelt, Gerjen H and Hilvering, Bart and Wouters, Kristiaan and van Staveren, Selma and Postma, Geert J and Vrisekoop, Nienke and Buydens, Lutgarde M C and Koenderman, Leo and Jansen, Jeroen J
Journal: Scientific reports (2020): 9716
Immunophenotyping of A20 haploinsufficiency by multicolor flow cytometry.
Authors: Kadowaki, Tomonori and Ohnishi, Hidenori and Kawamoto, Norio and Kadowaki, Saori and Hori, Tomohiro and Nishimura, Kenichi and Kobayashi, Chie and Shigemura, Tomonari and Ogata, Shohei and Inoue, Yuzaburo and Hiejima, Eitaro and Izawa, Kazushi and Matsubayashi, Tadashi and Matsumoto, Kazuaki and Imai, Kohsuke and Nishikomori, Ryuta and Ito, Shuichi and Kanegane, Hirokazu and Fukao, Toshiyuki
Journal: Clinical immunology (Orlando, Fla.) (2020): 108441
Authors: Kadowaki, Tomonori and Ohnishi, Hidenori and Kawamoto, Norio and Kadowaki, Saori and Hori, Tomohiro and Nishimura, Kenichi and Kobayashi, Chie and Shigemura, Tomonari and Ogata, Shohei and Inoue, Yuzaburo and Hiejima, Eitaro and Izawa, Kazushi and Matsubayashi, Tadashi and Matsumoto, Kazuaki and Imai, Kohsuke and Nishikomori, Ryuta and Ito, Shuichi and Kanegane, Hirokazu and Fukao, Toshiyuki
Journal: Clinical immunology (Orlando, Fla.) (2020): 108441
Post-induction Measurable Residual Disease Using Multicolor Flow Cytometry Is Strongly Predictive of Inferior Clinical Outcome in the Real-Life Management of Childhood T-Cell Acute Lymphoblastic Leukemia: A Study of 256 Patients.
Authors: Tembhare, Prashant R and Narula, Gaurav and Khanka, Twinkle and Ghogale, Sitaram and Chatterjee, Gaurav and Patkar, Nikhil V and Prasad, Maya and Badrinath, Yajamanam and Deshpande, Nilesh and Gudapati, Pratyusha and Verma, Shefali and Sanyal, Mahima and Kunjachan, Florence and Mangang, Gunit and Gujral, Sumeet and Banavali, Shripad and Subramanian, Papagudi G
Journal: Frontiers in oncology (2020): 577
Authors: Tembhare, Prashant R and Narula, Gaurav and Khanka, Twinkle and Ghogale, Sitaram and Chatterjee, Gaurav and Patkar, Nikhil V and Prasad, Maya and Badrinath, Yajamanam and Deshpande, Nilesh and Gudapati, Pratyusha and Verma, Shefali and Sanyal, Mahima and Kunjachan, Florence and Mangang, Gunit and Gujral, Sumeet and Banavali, Shripad and Subramanian, Papagudi G
Journal: Frontiers in oncology (2020): 577
Analysis of the immune status from peripheral whole blood with a single-tube multicolor flow cytometry assay.
Authors: Donaubauer, Anna-Jasmina and Becker, Ina and Rühle, Paul F and Fietkau, Rainer and Gaipl, Udo S and Frey, Benjamin
Journal: Methods in enzymology (2020): 389-415
Authors: Donaubauer, Anna-Jasmina and Becker, Ina and Rühle, Paul F and Fietkau, Rainer and Gaipl, Udo S and Frey, Benjamin
Journal: Methods in enzymology (2020): 389-415
Combination of multicolor flow cytometry for circulating lymphoma cells and tests for the RHOAG17V and IDH2R172 hot-spot mutations in plasma cell-free DNA as liquid biopsy for the diagnosis of angioimmunoblastic T-cell lymphoma.
Authors: Hayashida, Masahiko and Maekawa, Fumiyo and Chagi, Yoshinari and Iioka, Futoshi and Kobashi, Yoichiro and Watanabe, Mikio and Ohno, Hitoshi
Journal: Leukemia & lymphoma (2020): 1-10
Authors: Hayashida, Masahiko and Maekawa, Fumiyo and Chagi, Yoshinari and Iioka, Futoshi and Kobashi, Yoichiro and Watanabe, Mikio and Ohno, Hitoshi
Journal: Leukemia & lymphoma (2020): 1-10
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