ReadiLink™ Rapid trFluor™ Eu Antibody Labeling Kit *Microscale Optimized for Labeling 50 ug Antibody Per Reaction*

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<p>ReadiLink™ antibody labeling kits provide one of the most convenient ways to label antibodies in microscale. The kits only require two simple mixing steps without a purification step involved. Each ReadiLink™ antibody labeling kit provides all the essential components for performing two conjugation reactions. Each kit can be used to label monoclonal, polyclonal antibodies or other proteins with only two simple mixing steps.</p>
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Unit Size: Cat No: Price (USD): Qty:
2 Labelings 1300 $950


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

Ex/Em (nm)346/617
SolventDMSO
Storage Freeze (<-15 °C)
Minimize light exposure
Category Superior Labeling Dyes
trFluor Dyes and Kits
Related
Many biological compounds present in cells, serum or other biological fluids are naturally fluorescent, and thus the use of conventional, prompt fluorophores leads to serious limitations in assay sensitivity due to the high background caused by the autofluorescence of the biological molecules to be assayed. The use of long-lived fluorophores combined with time-resolved detection (a delay between excitation and emission detection) minimizes prompt fluorescence interferences. Our TR Fluor™ Eu probes enable time-resolved fluorometry (TRF) for the assays that require high sensitivity. These TR Fluor™ Eu probes have large Stokes shifts and extremely long emission half-lives when compared to more traditional fluorophores such as Alexa Fluor or cyanine dyes. Compared to the other TRF compounds, our TR Fluor™ Eu probes have relatively high stability, high emission yield and ability to be linked to biomolecules. Moreover, our TR Fluor™ Eu probes are insensitive to fluorescence quenching when conjugated to biological polymers such as antibodies.




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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.

1. Prepare protein solution (Solution A):

For labeling 50 µg protein (assuming the target protein concentration is 1 mg/mL), mix 1.5 µL (3% of the total reaction volume) of Reaction Buffer (Component B) with 50 µL of the target protein solution.

Note 1. If you have a difference protein concentration, adjust the protein volume accordingly to make ~50 µg protein available for your labeling reaction.

Note 2: For labeling 100 µg protein (assuming the target protein concentration is 1 mg/mL), mix 3 µL (3% of the total reaction volume) of Reaction Buffer (Component B) with 100 µL of the target protein solution.

Note 3: The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4; If the protein is dissolved in glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, or use Amicon Ultra-0.5, Ultracel-10 Membrane, 10 kDa (cat# UFC501008 from Millipore) to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation.

Note 4: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.

Note 5: The conjugation efficiency is significantly reduced if the protein concentration is less than 1 mg/mL. For optimal labeling efficiency the final protein concentration range of 1-2 mg/mL is recommended.

2. Run conjugation reaction:

2.1    Add the protein solution (Solution A) to ONE vial of labeling dye (Component A), and mix them well by repeatedly pipetting for a few times or vortex the vial for a few seconds.

Note: Use both vials (Component A) of labeling dye to label 100 µg protein by dividing the 100 µg protein into 2x50 µg protein and reacting each 50 µg protein with one vial of labeling dye. Combine two vials for the next step.

2.2    Keep the conjugation reaction mixture at room temperature for 30 - 60 minutes.

Note: The conjugation reaction mixture can be rotated or shaken for longer time if desired.

3. Prepare spin column for sample purification:

3.1    Invert the Spin Column (Component C) several times to resuspend the settled gel and remove any bubbles.

3.2    Snap off the tip and place the column in the Washing Tube (2 mL, Component D). Remove the cap to allow the excess packing buffer to drain by gravity to the top of the gel bed. If column does not begin to flow, push cap back into column and remove it again to start the flow. Discard the drained buffer, and then place the column back into the Washing Tube. However, centrifuge immediately if the column is placed into a 12 x 75 mm test tube (not provided).

3.3    Centrifuge for 1 min in a swinging bucket centrifuge at 1,000 x g (see Centrifugation Notes section) to remove the packing buffer. Discard the buffer.

3.4    Apply 1-2 mL 1X PBS (pH 7.2-7.4) to the column. After each application of PBS, let the buffer drain out by gravity, or centrifuge the column for 1 min to remove the buffer. Discard the buffer from the collection tube. Repeat this process for 3-4 times.

3.5    Centrifuge for 2 minutes in a swinging bucket centrifuge at 1,000 x g (see Centrifugation Notes section) to remove the packing buffer. Discard the buffer.

 

4. Purify the conjugation:

4.1    Place the column (from Step 3.5) in a clean Collecting Tube (1.5 mL, Component E). Carefully load the sample (50–100 μL) directly to the center of the column.

4.2    After loading the sample, add 1X PBS (pH 7.2-7.4) to make the total volume of 110 μL. Centrifuge the column for 5 min at 1,000 x g, and collect the solution that contains the desired dye-labeled protein.






References & Citations

Development of a time-resolved fluorescence resonance energy transfer assay for cyclin-dependent kinase 4 and identification of its ATP-noncompetitive inhibitors
Authors: Lo MC, Ngo R, Dai K, Li C, Liang L, Lee J, Emkey R, Eksterowicz J, Ventura M, Young SW, Xiao SH.
Journal: Anal Biochem (2012): 368

Time-Resolved Fluorescence Resonance Energy Transfer as a Versatile Tool in the Development of Homogeneous Cellular Kinase Assays
Authors: Saville L, Spais C, Mason JL, Albom MS, Murthy S, Meyer SL, Ator MA, Angeles TS, Husten J.
Journal: Assay Drug Dev Technol. (2012)

A homogeneous single-label time-resolved fluorescence cAMP assay
Authors: Martikkala E, Rozwandowicz-Jansen A, Hanninen P, Petaja-Repo U, Harma H.
Journal: J Biomol Screen (2011): 356

Homogeneous time-resolved fluorescence-based assay to screen for ligands targeting the growth hormone secretagogue receptor type 1a
Authors: Leyris JP, Roux T, Trinquet E, Verdie P, Fehrentz JA, Oueslati N, Douzon S, Bourrier E, Lamarque L, Gagne D, Galleyrand JC, M'Kadmi C, Martinez J, Mary S, Baneres JL, Marie J.
Journal: Anal Biochem (2011): 253

Oligomerization of the serotonin(1A) receptor in live cells: a time-resolved fluorescence anisotropy approach
Authors: Paila YD, Kombrabail M, Krishnamoorthy G, Chattopadhyay A.
Journal: J Phys Chem B (2011): 11439

Time-resolved fluorescence resonance energy transfer (TR-FRET) to analyze the disruption of EGFR/HER2 dimers: a new method to evaluate the efficiency of targeted therapy using monoclonal antibodies
Authors: Gaborit N, Larbouret C, Vallaghe J, Peyrusson F, Bascoul-Mollevi C, Crapez E, Azria D, Chardes T, Poul MA, Mathis G, Bazin H, Pelegrin A.
Journal: J Biol Chem (2011): 11337

A time-resolved fluorescence-resonance energy transfer assay for identifying inhibitors of hepatitis C virus core dimerization
Authors: Kota S, Scampavia L, Spicer T, Beeler AB, Takahashi V, Snyder JK, Porco JA, Hodder P, Strosberg AD.
Journal: Assay Drug Dev Technol (2010): 96

Ligand regulation of the quaternary organization of cell surface M3 muscarinic acetylcholine receptors analyzed by fluorescence resonance energy transfer (FRET) imaging and homogeneous time-resolved FRET
Authors: Alvarez-Curto E, Ward RJ, Pediani JD, Milligan G.
Journal: J Biol Chem (2010): 23318

Steady-state and time-resolved fluorescence quenching with transition metal ions as short-distance probes for protein conformation
Authors: Posokhov YO, Kyrychenko A, Ladokhin AS.
Journal: Anal Biochem (2010): 284

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs
Authors: Visser AJ, Laptenok SP, Visser NV, van Hoek A, Birch DJ, Brochon JC, Borst JW.
Journal: Eur Biophys J (2010): 241


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