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iFluor® 488 succinimidyl ester

HeLa cells were stained with rabbit anti-tubulin followed by iFluor 488 goat anti-rabbit IgG (H+L), and nuclei were stained with Nuclear Red DCS1.
HeLa cells were stained with rabbit anti-tubulin followed by iFluor 488 goat anti-rabbit IgG (H+L), and nuclei were stained with Nuclear Red DCS1.
Flow cytometry analysis of PBMC stained with iFluor® 488 anti-human CD24 *HI45* conjugate. The fluorescence signal was monitored using an Aurora flow cytometer in the iFluor® 488 specific B2-A channel.
Fluorescence In Situ Hybridization of Fluorescein and iFluor® 488 labelled Telomere probes in metaphase HeLa cells.
HeLa cells were incubated with (Tubulin+) or without (Tubulin-) mouse anti-tubulin followed by iFluor® 488 goat anti-mouse IgG conjugate (Green, Left) or Alexa Fluor® 488 goat anti-mouse IgG conjugate (Green, Right), respectively. Cell nuclei were stained with Hoechst 33342 (Blue).
Flow cytometric analysis of Alexa Fluor® 488 or iFluor® 488 anti-human CD4 on human lymphocytes. PBMC cells were stained with 0.5 ug Alexa Fluor® 488 anti-human CD4  or  0.5 ug iFluor® 488 anti-human CD4 in each test.  Flow cytometric analysis was performed on a ACEA flow cytometry system.
Ordering information
Price ()
Catalog Number1023
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight945.07
SolventDMSO
Spectral properties
Correction Factor (260 nm)0.21
Correction Factor (280 nm)0.11
Extinction coefficient (cm -1 M -1)750001
Excitation (nm)491
Emission (nm)516
Quantum yield0.91
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501
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iFluor® 488 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 514 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 532 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 555 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 594 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 633 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 647 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 680 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 700 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 750 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 790 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 350 goat anti-rabbit IgG (H+L)
iFluor® 405 goat anti-rabbit IgG (H+L)
iFluor® 488 goat anti-rabbit IgG (H+L)
iFluor® 514 goat anti-rabbit IgG (H+L)
iFluor® 532 goat anti-rabbit IgG (H+L)
iFluor® 555 goat anti-rabbit IgG (H+L)
iFluor® 594 goat anti-rabbit IgG (H+L)
iFluor® 633 goat anti-rabbit IgG (H+L)
iFluor® 647 goat anti-rabbit IgG (H+L)
iFluor® 680 goat anti-rabbit IgG (H+L)
iFluor® 700 goat anti-rabbit IgG (H+L)
iFluor® 750 goat anti-rabbit IgG (H+L)
iFluor® 790 goat anti-rabbit IgG (H+L)
iFluor® 350 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 405 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 488 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 514 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 532 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 555 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 594 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 633 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
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iFluor® 680 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 700 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
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iFluor® 350-streptavidin conjugate
iFluor® 405-streptavidin conjugate
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iFluor® 800 acid
iFluor® 800 maleimide
iFluor® 810 acid
iFluor® 810 maleimide
iFluor® 820 acid
iFluor® 820 maleimide
iFluor® 860 acid
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iFluor® 546 goat anti-mouse IgG (H+L)
iFluor® 546 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 568 goat anti-mouse IgG (H+L)
iFluor® 568 goat anti-mouse IgG (H+L) *Cross Adsorbed*
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iFluor® 430 maleimide
iFluor® 568 maleimide
iFluor® 633 maleimide
iFluor® 450 maleimide
iFluor® 350 Styramide *Superior Replacement for Alexa Fluor 350 tyramide*
iFluor® 488 Styramide *Superior Replacement for Alexa Fluor 488 tyramide and Opal 520*
iFluor® 546 Styramide *Superior Replacement for Alexa Fluor 546 tyramide*
iFluor® 555 Styramide *Superior Replacement for Alexa Fluor 555 tyramide and Opal 570*
iFluor® 568 Styramide *Superior Replacement for Alexa Fluor 568 tyramide*
iFluor® 594 Styramide *Superior Replacement for Alexa Fluor 594 tyramide*
iFluor® 647 Styramide *Superior Replacement for Alexa Fluor 647 tyramide*
iFluor® 680 Styramide *Superior Replacement for Alexa Fluor 680 tyramide and Opal 690*
iFluor® 700 Styramide *Superior Replacement for Alexa Fluor 700 tyramide*
iFluor® 750 Styramide *Superior Replacement for Alexa Fluor 750 tyramide*
iFluor® 790 Styramide *Superior Replacement for Alexa Fluor 790 tyramide*
iFluor® 555 Tyramide
iFluor® 647 Tyramide
iFluor® 350 PSA™ Imaging Kit with Goat Anti-Rabbit IgG
iFluor® 488 PSA™ Imaging Kit with Goat Anti-Rabbit IgG
iFluor® 555 PSA™ Imaging Kit with Goat Anti-Rabbit IgG
iFluor® 594 PSA™ Imaging Kit with Goat Anti-Rabbit IgG
iFluor® 647 PSA™ Imaging Kit with Goat Anti-Rabbit IgG
iFluor 350™ PSA™ Imaging Kit with Goat Anti-Mouse IgG
iFluor 488™ PSA™ Imaging Kit with Goat Anti-Mouse IgG
iFluor 555™ PSA™ Imaging Kit with Goat Anti-Mouse IgG
iFluor 594™ PSA™ Imaging Kit with Goat Anti-Mouse IgG
iFluor 647™ PSA™ Imaging Kit with Goat Anti-Mouse IgG
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iFluor® 800-streptavidin conjugate
iFluor® 820-streptavidin conjugate
iFluor® 840-streptavidin conjugate
iFluor® 860-streptavidin conjugate
iFluor® 488-Wheat Germ Agglutinin (WGA) Conjugate
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iFluor® 488-Concanavalin A Conjugate
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iFluor® 568 Tyramide
iFluor® 594 Tyramide
iFluor® 460 maleimide
iFluor® 488 TCO
iFluor® 555 TCO
iFluor® 594 TCO
iFluor® 647 TCO
iFluor® 488 Tetrazine
iFluor® 555 Tetrazine
iFluor® 594 Tetrazine
iFluor® 647 Tetrazine
iFluor® 440-dUTP *1 mM in Tris Buffer (pH 7.5)*
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iFluor®488-dUTP *1 mM in Tris Buffer (pH 7.5)*
iFluor® 450 Styramide *Superior Replacement for Opal Polaris 480*
iFluor® 633 tyramide
iFluor® 514 Styramide *Superior Replacement for Opal 540*
iFluor® 532 Styramide
iFluor® 633 Styramide *Superior Replacement for Opal 650*
iFluor® 440 Styramide
iFluor® 460 Styramide
iFluor® 610 Styramide
iFluor® 660 Styramide
iFluor® 405 Styramide
iFluor®488-PEG12-dUTP *1 mM in Tris Buffer (pH 7.5)*
iFluor®555-PEG12-dUTP *1 mM in Tris Buffer (pH 7.5)*
iFluor®647-PEG12-dUTP *1 mM in Tris Buffer (pH 7.5)*
iFluor® 800 goat anti-mouse IgG (H+L)
iFluor® 800 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 810 goat anti-mouse IgG (H+L)
iFluor® 810 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 820 goat anti-mouse IgG (H+L)
iFluor® 820 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 840 goat anti-mouse IgG (H+L)
iFluor® 840 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 860 goat anti-mouse IgG (H+L)
iFluor® 860 goat anti-mouse IgG (H+L) *Cross Adsorbed*
iFluor® 800 goat anti-rabbit IgG (H+L)
iFluor® 800 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 810 goat anti-rabbit IgG (H+L)
iFluor® 810 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 820 goat anti-rabbit IgG (H+L)
iFluor® 820 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 840 goat anti-rabbit IgG (H+L)
iFluor® 840 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 860 goat anti-rabbit IgG (H+L)
iFluor® 860 goat anti-rabbit IgG (H+L) *Cross Adsorbed*
iFluor® 430 Tyramide *Superior Replacement for Opal 480*
iFluor® 450 Tyramide *Superior Replacement for Opal 480*
iFluor® 546 maleimide
iFluor® 840 maleimide
iFluor® 770 maleimide
iFluor® 780 maleimide
iFluor® Ultra 594 succinimidyl ester
iFluor® Ultra 647 succinimidyl ester
iFluor® Ultra 750 succinimidyl ester
iFluor® 830 acid
iFluor® 830 maleimide
iFluor™ 405 azide
iFluor® 514 maleimide
iFluor® 660 maleimide
iFluor® 670 maleimide
Show More (223)

OverviewpdfSDSpdfProtocol


Molecular weight
945.07
Correction Factor (260 nm)
0.21
Correction Factor (280 nm)
0.11
Extinction coefficient (cm -1 M -1)
750001
Excitation (nm)
491
Emission (nm)
516
Quantum yield
0.91
Although FITC is still the most popular fluorescent labeling dye for preparing green fluorescent bioconjugates, there are certain limitations with FITC, such as severe photobleaching for microscope imaging and pH-sensitive fluorescence. Protein conjugates prepared with iFluor® 488 dyes are far superior to conjugates of fluorescein derivatives such as FITC. iFluor® 488 conjugates are significantly brighter than fluorescein conjugates and are much more photostable. Additionally, the fluorescence of iFluor® 488 is not affected by pH (4-10). This pH insensitivity is a major improvement over fluorescein, which emits its maximum fluorescence only at pH above 9. iFluor® 488 SE dye is reasonably stable and shows good reactivity and selectivity with protein amino groups. This iFluor® 488 has spectral properties and reactivity similar to Alexa Fluor® 488 NHS ester ( Alexa Fluor® is the trademark of Invitrogen).

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.

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. iFluor™ 488 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ 350 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 iFluor™ 350 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.


Run conjugation reaction
  1. 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.
  2. 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.
  1. Prepare Sephadex G-25 column according to the manufacture instruction.
  2. Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
  3. Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
  4. 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. 

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of iFluor® 488 succinimidyl ester to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM105.812 µL529.061 µL1.058 mL5.291 mL10.581 mL
5 mM21.162 µL105.812 µL211.625 µL1.058 mL2.116 mL
10 mM10.581 µL52.906 µL105.812 µL529.061 µL1.058 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Correction Factor (260 nm)0.21
Correction Factor (280 nm)0.11
Extinction coefficient (cm -1 M -1)750001
Excitation (nm)491
Emission (nm)516
Quantum yield0.91

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yieldCorrection Factor (260 nm)Correction Factor (280 nm)
iFluor® 350 succinimidyl ester3454502000010.9510.830.23
iFluor® 405 succinimidyl ester4034273700010.9110.480.77
iFluor® 514 succinimidyl ester5115277500010.8310.2650.116
iFluor® 532 succinimidyl ester5375609000010.6810.260.16
iFluor® 555 succinimidyl ester55757010000010.6410.230.14
iFluor® 594 succinimidyl ester58860418000010.5310.050.04
iFluor® 633 succinimidyl ester64065425000010.2910.0620.044
iFluor® 647 succinimidyl ester65667025000010.2510.030.03
iFluor® 660 succinimidyl ester66367825000010.2610.070.08
iFluor® 680 succinimidyl ester68470122000010.2310.0970.094
iFluor® 700 succinimidyl ester69071322000010.2310.090.04
iFluor® 750 succinimidyl ester75777927500010.1210.0440.039
iFluor® 610 succinimidyl ester61062811000010.8510.320.49
iFluor® 710 succinimidyl ester71773919000010.6010.120.07
iFluor® 790 succinimidyl ester78781225000010.1310.10.09
iFluor® 800 succinimidyl ester80182025000010.1110.030.08
iFluor® 810 succinimidyl ester81182225000010.0510.090.15
iFluor® 820 succinimidyl ester8228502500001-0.110.16
iFluor® 860 succinimidyl ester8538782500001-0.10.14
iFluor® 546 succinimidyl ester54155710000010.6710.250.15
iFluor® 568 succinimidyl ester56858710000010.5710.340.15
iFluor® 430 succinimidyl ester4334984000010.7810.680.3
iFluor® 450 succinimidyl ester4515024000010.8210.450.27
iFluor® 840 succinimidyl ester8368792000001-0.20.09
iFluor® 560 succinimidyl ester56057112000010.5710.04820.069
iFluor® 670 succinimidyl ester67168220000010.5510.030.033
iFluor® 460 succinimidyl ester468493800001~0.810.980.46
iFluor® 440 succinimidyl ester4344804000010.6710.3520.229
iFluor® 665 succinimidyl ester667692110,00010.2210.120.09
iFluor® 690 succinimidyl ester68570422000010.3010.090.06
iFluor® 720 succinimidyl ester71674024000010.1410.150.13
iFluor® 740 succinimidyl ester74276422500010.2010.160.16
iFluor® 597 succinimidyl ester59861810000010.710.3350.514
iFluor® 770 succinimidyl ester77779725000010.160.090.08
iFluor® 780 succinimidyl ester78480825000010.1610.130.12
iFluor® 570 succinimidyl ester56057112000010.5810.0480.069
iFluor® 830 succinimidyl ester830867----
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Citations


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Journal: Nanomaterials (2021): 264
Essential role of smooth muscle Rac1 in severe asthma-associated airway remodelling
Authors: Dilasser, Florian and Rose, Lindsay and Hassoun, Dorian and Klein, Martin and Rousselle, Morgane and Brosseau, Carole and Guignabert, Christophe and Taill{\'e}, Camille and Dombret, Marie Christine and Di Candia, Leonarda and others,
Journal: Thorax (2021): 326--334
miR-19a-3p Facilitates Lung Adenocarcinoma Cell Phenotypes by Inhibiting TEK
Authors: Peng, Tao and Yang, Fan and Sun, Zhanwen and Yan, Jie
Journal: Cancer Biotherapy &Radiopharmaceuticals (2021)
Self-assembled organic nanomedicine enables ultrastable photo-to-heat converting theranostics in the second near-infrared biowindow
Authors: Xiang, Huijing and Zhao, Lingzhi and Yu, Luodan and Chen, Hongzhong and Wei, Chenyang and Chen, Yu and Zhao, Yanli
Journal: Nature communications (2021): 1--12
CD95/Fas protects triple negative breast cancer from anti-tumor activity of NK cells
Authors: Qadir, Abdul S and Gu{\'e}gan, Jean Philippe and Ginestier, Christophe and Chaibi, Assia and Bessede, Alban and Charafe-Jauffret, Emmanuelle and Macario, Manon and Lavou{\'e}, Vincent and de la Motte Rouge, Thibault and Law, Calvin and others,
Journal: Iscience (2021): 103348
CaMKII activation persistently segregates postsynaptic proteins via liquid phase separation
Authors: Hosokawa, Tomohisa and Liu, Pin-Wu and Cai, Qixu and Ferreira, Joana S and Levet, Florian and Butler, Corey and Sibarita, Jean-Baptiste and Choquet, Daniel and Groc, Laurent and Hosy, Eric and others,
Journal: Nature Neuroscience (2021): 777--785
Biofunctional Silk Kirigami With Engineered Properties
Authors: Pradhan, Sayantan and Ventura, Leonardo and Agostinacchio, Francesca and Xu, Meng and Barbieri, Ettore and Motta, Antonella and Pugno, Nicola M and Yadavalli, Vamsi K
Journal: ACS Applied Materials \& Interfaces (2020): 12436--12444
Bile salt--dependent lipase promotes the barrier integrity of Caco-2 cells by activating Wnt/$\beta$-catenin signaling via LRP6 receptor
Authors: Qiu, Yaqi and Zhou, Jiefei and Zhang, Dandan and Song, Huanlei and Qian, Linxi
Journal: Cell and Tissue Research (2020): 1--16
TEK Suppresses Lung Adenocarcinoma Cell Phenotypes by Interacting with miR-19a-3p
Authors: Peng, Tao and Yang, Fan and Sun, Zhanwen and Yan, Jie
Journal: (2020)
CaMKII activation triggers persistent formation and segregation of postsynaptic liquid phase
Authors: Hosokawa, Tomohisa and Liu, Pin-Wu and Cai, Qixu and Ferreira, Joana S and Levet, Florian and Butler, Corey and Sibarita, Jean-Baptiste and Choquet, Daniel and Groc, Laurent and Hosy, Eric and others,
Journal: bioRxiv (2020)

References


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Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers
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Journal: Zhen Ci Yan Jiu (2010): 433
Photoactivatable and photoconvertible fluorescent probes for protein labeling
Authors: Maurel D, Banala S, Laroche T, Johnsson K.
Journal: ACS Chem Biol (2010): 507
Novel Alexa Fluor-488 labeled antagonist of the A(2A) adenosine receptor: Application to a fluorescence polarization-based receptor binding assay
Authors: Kecskes M, Kumar TS, Yoo L, Gao ZG, Jacobson KA.
Journal: Biochem Pharmacol (2010): 506