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iFluor® A7 SE

Human peripheral blood lymphocytes were stained with 5 uL (0.25 ug) APC-A7 Anti-Human HLA-DR (solid line) or 0.25 ug APC-A7 Mouse IgG2a isotype control (dashed line). The L243 antibody reacts with a member of the human MHC Class II antigens, HLA-DR. The HLA-DR antigen is expressed on B lymphocytes, activated T lymphocytes, activated NK cells, monocytes, macrophages, other antigen presenting cells and progenitor cells. The L243 antibody is specific to an epitope on the alpha subunit of the heterodimeric HLA-DR protein and binds a different epitope than the LN3 antibody clone. It does not cross-react with HLA-DP or HLA-DQ. This antibody is reported to be cross-reactive with non-human primates including Chimpanzee, Cynomolgus, Rhesus and Baboon. Please choose the appropriate format for each application.
Human peripheral blood lymphocytes were stained with 5 uL (0.25 ug) APC-A7 Anti-Human HLA-DR (solid line) or 0.25 ug APC-A7 Mouse IgG2a isotype control (dashed line). The L243 antibody reacts with a member of the human MHC Class II antigens, HLA-DR. The HLA-DR antigen is expressed on B lymphocytes, activated T lymphocytes, activated NK cells, monocytes, macrophages, other antigen presenting cells and progenitor cells. The L243 antibody is specific to an epitope on the alpha subunit of the heterodimeric HLA-DR protein and binds a different epitope than the LN3 antibody clone. It does not cross-react with HLA-DP or HLA-DQ. This antibody is reported to be cross-reactive with non-human primates including Chimpanzee, Cynomolgus, Rhesus and Baboon. Please choose the appropriate format for each application.
Ordering information
Price ()
Catalog Number1039
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 weight1288.58
SolventDMSO
Spectral properties
Absorbance (nm)759
Correction Factor (260 nm)0.03
Correction Factor (280 nm)0.03
Extinction coefficient (cm -1 M -1)275000
Excitation (nm)762
Emission (nm)782
Quantum yield0.1
Storage, safety and handling
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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Show More (265)

OverviewpdfSDSpdfProtocol


Molecular weight
1288.58
Absorbance (nm)
759
Correction Factor (260 nm)
0.03
Correction Factor (280 nm)
0.03
Extinction coefficient (cm -1 M -1)
275000
Excitation (nm)
762
Emission (nm)
782
Quantum yield
0.1
AAT Bioquest's iFluor® A7 dye was developed for labeling proteins, particularly antibodies. iFluor® A7 has a fluorescence excitation and emission maxima of ~762 nm and ~782 nm respectively, with good photostability. The APC-iFluor® A7 tandem is an excellent replacement for BD's APC-H7. Our in-house comparable studies indicated that the APC-iFluor® A7 tandem demonstrated comparable fluorescence properties to the APC-H7 tandem. The A7 SE dye is reasonably stable and shows good reactivity and selectivity with proteins.

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™ A7 SE stock solution (Solution B)
Add anhydrous DMSO into the vial of iFluor™ A7 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™ A7 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® A7 SE 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 mM77.605 µL388.024 µL776.048 µL3.88 mL7.76 mL
5 mM15.521 µL77.605 µL155.21 µL776.048 µL1.552 mL
10 mM7.76 µL38.802 µL77.605 µL388.024 µL776.048 µL

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

Absorbance (nm)759
Correction Factor (260 nm)0.03
Correction Factor (280 nm)0.03
Extinction coefficient (cm -1 M -1)275000
Excitation (nm)762
Emission (nm)782
Quantum yield0.1

Citations


View all 3 citations: Citation Explorer
Deep Sequencing Analysis of the Eha-Regulated Transcriptome of Edwardsiella tarda Following Acidification
Authors: Gao, D and Liu, N and Li, Y and Zhang, Y and Liu, G and others, undefined
Journal: Metabolomics (Los Angel) (2017): 2153--0769
Suramin inhibits cullin-RING E3 ubiquitin ligases
Authors: Wu, Kenneth and Chong, Robert A and Yu, Qing and Bai, Jin and Spratt, Donald E and Ching, Kevin and Lee, Chan and Miao, Haibin and Tappin, Inger and Hurwitz, Jerard and others, undefined
Journal: Proceedings of the National Academy of Sciences (2016): E2011--E2018
Glycosaminoglycan mimicry by COAM reduces melanoma growth through chemokine induction and function
Authors: Piccard, Helene and Berghmans, Nele and Korpos, Eva and Dillen, Chris and Aelst, Ilse Van and Li, S and ra , undefined and Martens, Erik and Liekens, S and ra , undefined and Noppen, Sam and Damme, Jo Van and others, undefined
Journal: International Journal of Cancer (2012): E425--E436

References


View all 49 references: Citation Explorer
Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based beta-lactam probes
Authors: Sadhu KK, Mizukami S, Watanabe S, Kikuchi K.
Journal: Mol Biosyst (2011): 1766
Visualizing dengue virus through Alexa Fluor labeling
Authors: Zhang S, Tan HC, Ooi EE.
Journal: J Vis Exp. (2011)
Fluorescent "Turn-on" system utilizing a quencher-conjugated peptide for specific protein labeling of living cells
Authors: Arai S, Yoon SI, Murata A, Takabayashi M, Wu X, Lu Y, Takeoka S, Ozaki M.
Journal: Biochem Biophys Res Commun (2011): 211
Neuroanatomical basis of clinical joint application of "Jinggu" (BL 64, a source-acupoint) and "Dazhong" (KI 4, a Luo-acupoint) in the rat: a double-labeling study of cholera toxin subunit B conjugated with Alexa Fluor 488 and 594
Authors: Cui JJ, Zhu XL, Ji CF, Jing XH, Bai WZ.
Journal: Zhen Ci Yan Jiu (2011): 262
Simultaneous detection of virulence factors from a colony in diarrheagenic Escherichia coli by a multiplex PCR assay with Alexa Fluor-labeled primers
Authors: Kuwayama M, Shigemoto N, Oohara S, Tanizawa Y, Yamada H, Takeda Y, Matsuo T, Fukuda S.
Journal: J Microbiol Methods (2011): 119
Alexa Fluor 546-ArIB[V11L;V16A] is a potent ligand for selectively labeling alpha 7 nicotinic acetylcholine receptors
Authors: Hone AJ, Whiteaker P, Mohn JL, Jacob MH, McIntosh JM.
Journal: J Neurochem (2010): 994
Asymmetric trimethine 3H-indocyanine dyes: efficient synthesis and protein labeling
Authors: Song F, Wang L, Qiao X, Wang B, Sun S, Fan J, Zhang L, Peng X.
Journal: Org Biomol Chem (2010): 4249
Neuroanatomical characteristics of acupoint "Chengshan" (BL 57) in the rat: a cholera toxin subunit B conjugated with Alexa Fluor 488 method study
Authors: Zhu XL, Bai WZ, Wu FD, Jiang J, Jing XH.
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