Actively helping customers, employees and the global community during the coronavirus SARS-CoV-2 outbreak.  Learn more >>

ReadiLink™ xtra Rapid iFluor® 488 Antibody Labeling Kit *BSA-Compatible*

Immunofluorescence staining of tubulin in HeLa cells. HeLa cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and blocked. Cells were then incubated with mouse anti-tubulin antibody (1ug/ml) and stained with a goat anti-mouse IgG conjugate (5ug/ml) labeled using different methods:
<ol>
<li>GXM IgG-iFluor®488 (Cat#16448)</li>
<li> 50ug GXM IgG in PBS with ReadiLink™ Rapid iFluor® 488 Antibody Labeling Kit (Cat#1255)</li>
<li>50ug GXM IgG in PBS + 0.05% BSA with ReadiLink™ Rapid iFluor® 488 Antibody Labeling Kit (Cat#1255)</li>
<li>50ug GXM IgG in PBS + 0.05% BSA with  ReadiLink™ xtra Rapid iFluor® 488 Antibody Labeling Kit (Cat#1955)</li>
<li>50ug GXM IgG in PBS + 0.5% BSA with ReadiLink™ xtra Rapid iFluor® 488 Antibody Labeling Kit (Cat#1955)</li>
<li>GXM IgG-Alexa Fluor 488 (Vendor J)</li>
</ol>
Immunofluorescence staining of tubulin in HeLa cells. HeLa cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and blocked. Cells were then incubated with mouse anti-tubulin antibody (1ug/ml) and stained with a goat anti-mouse IgG conjugate (5ug/ml) labeled using different methods:
<ol>
<li>GXM IgG-iFluor®488 (Cat#16448)</li>
<li> 50ug GXM IgG in PBS with ReadiLink™ Rapid iFluor® 488 Antibody Labeling Kit (Cat#1255)</li>
<li>50ug GXM IgG in PBS + 0.05% BSA with ReadiLink™ Rapid iFluor® 488 Antibody Labeling Kit (Cat#1255)</li>
<li>50ug GXM IgG in PBS + 0.05% BSA with  ReadiLink™ xtra Rapid iFluor® 488 Antibody Labeling Kit (Cat#1955)</li>
<li>50ug GXM IgG in PBS + 0.5% BSA with ReadiLink™ xtra Rapid iFluor® 488 Antibody Labeling Kit (Cat#1955)</li>
<li>GXM IgG-Alexa Fluor 488 (Vendor J)</li>
</ol>
Flow cytometry analysis of HL-60 cells stained with 1 µg/mL Mouse IgG control (Green) or with 1 µg/mL Anti-Human CD45-iFluor® 488 (Red) prepared using the ReadiLink™ xtra Rapid iFluor® 488 Antibody Labeling Kit (Cat No. 1955). The fluorescence signal was monitored using an ACEA NovoCyte flow cytometer in the FITC channel.
Immunofluorescence staining of tubulin in HeLa cells. HeLa cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and blocked. Cells were then incubated with mouse anti-tubulin antibody and stained with a goat anti-mouse IgG labeled using the ReadiLink™ xtra Rapid iFluor® 488 Antibody Labeling Kit (Cat No. 1955).
Ordering information
Price ()
Catalog Number1955
Unit Size
Find Distributor
Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
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
UNSPSC12171501

OverviewpdfSDSpdfProtocol


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
ReadiLink™ xtra rapid antibody labeling kits require essentially only 2 simple mixing steps without a column purification needed. Preactivated iFluor® 488 used in this ReadiLink™ kit is quite stable and shows good reactivity and selectivity with antibodies. The kit has all the essential components for labeling ~2x50 ug antibody. Each of the two vials of preactivated iFluor® 488 dye provided in the kit is optimized for labeling ~50 µg antibody. ReadiLink™ xtra iFluor® 488 rapid antibody labeling kit provides a convenient and robust method to label monoclonal and polyclonal antibodies with the bright green fluorescent iFluor® 488 fluorophore. AAT Bioquest's iFluor® dyes are optimized for labeling proteins, in particular, antibodies. These dyes are bright, photostable and have minimal quenching on proteins. They can be well excited by the major laser lines of fluorescence instruments (e.g., 350, 405, 488, 555 and 633 nm).

 

readilinkworkflow

 

Figure 1. Overview of the ReadiLink™ xtra Rapid Antibody Labeling protocol. In just two simple steps, and with no purification necessary, covalently label microgram amounts of antibodies in under an hour.

Components


Component A: Preactivated iFluor™ 4882 vials
Component B: Reaction Buffer1 vial (20 µL)
Component C: TQ™-Dyed Quench Buffer1 vial (20 µL)

Example protocol


AT A GLANCE

Important
Warm all the components and centrifuge the vials briefly before opening, and immediately prepare the required solutions before starting your conjugation. The following protocol is for recommendation.

PREPARATION OF WORKING SOLUTION

Protein working solution (Solution A)
For labeling 50 µg of protein (assuming the target protein concentration is 1 mg/mL), mix 5 µL (10% of the total reaction volume) of Reaction Buffer (Component B) with 50 µL of the target protein solution.
Note     If you have a different protein concentration, adjust the protein volume accordingly to make ~50 µg of protein available for your labeling reaction.
Note     For labeling 100 µg of protein (assuming the target protein concentration is 1 mg/mL), mix 10 µL (10% of the total reaction volume) of Reaction Buffer (Component B) with 100 µL of the target protein solution.
Note     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     Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) with 0.1 to 0.5 % will be labeled well.
Note     For optimal labeling efficiency, a final protein concentration range of 1 - 2 mg/mL is recommended, with a significantly reduced conjugation efficiency at less than 1 mg/mL.

SAMPLE EXPERIMENTAL PROTOCOL

Run conjugation reaction
  1. Add the protein working 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     If labeling 100 µg of protein, use both vials (Component A) of labeling dye by dividing the 100 µg of protein into 2 x 50 µg of protein and reacting each 50 µg of protein with one vial of labeling dye. Then combine both vials for the next step.
  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. 

Stop Conjugation reaction
  1. Add 5 µL (for 50 µg protein) or 10 µL (for 100 µg protein) which is 10% of the total reaction volume of TQ™-Dyed Quench Buffer (Component C) into the conjugation reaction mixture; mix well.
  2. Incubate at room temperature for 10 minutes. The labeled protein (antibody) is now ready to use. 

Storage of Protein Conjugate
The protein conjugate should be stored at > 0.5 mg/mL in the presence of a carrier protein (e.g., 0.1% bovine serum albumin). For longer storage, the protein conjugates could be lyophilized or divided into single-used aliquots and stored at ≤ –20 °C.

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

References


View all 27 references: Citation Explorer
Author Correction: A dynamic three-step mechanism drives the HIV-1 pre-fusion reaction.
Authors: Iliopoulou, Maro and Nolan, Rory and Alvarez, Luis and Watanabe, Yasunori and Coomer, Charles A and Jakobsdottir, G Maria and Bowden, Thomas A and Padilla-Parra, Sergi
Journal: Nature structural & molecular biology (2019): 526
Intracellular in situ labeling of TiO2 nanoparticles for fluorescence microscopy detection.
Authors: Brown, Koshonna and Thurn, Ted and Xin, Lun and Liu, William and Bazak, Remon and Chen, Si and Lai, Barry and Vogt, Stefan and Jacobsen, Chris and Paunesku, Tatjana and Woloschak, Gayle E
Journal: Nano research (2018): 464-476
Effect of Fluorescent Labels on Peptide and Amino Acid Sample Dimensionality in Two Dimensional nLC × μFFE Separations.
Authors: Geiger, Matthew and Bowser, Michael T
Journal: Analytical chemistry (2016): 2177-87
Probing minority population of antibiotic-resistant bacteria.
Authors: Huang, Tianxun and Zheng, Yan and Yan, Ya and Yang, Lingling and Yao, Yihui and Zheng, Jiaxin and Wu, Lina and Wang, Xu and Chen, Yuqing and Xing, Jinchun and Yan, Xiaomei
Journal: Biosensors & bioelectronics (2016): 323-330
Label-free imaging of gelatin-containing hydrogel scaffolds.
Authors: Liang, Yajie and Bar-Shir, Amnon and Song, Xiaolei and Gilad, Assaf A and Walczak, Piotr and Bulte, Jeff W M
Journal: Biomaterials (2015): 144-50
Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.
Authors: Liao-Chan, Sindy and Daine-Matsuoka, Barbara and Heald, Nathan and Wong, Tiffany and Lin, Tracey and Cai, Allen G and Lai, Michelle and D'Alessio, Joseph A and Theunissen, Jan-Willem
Journal: PloS one (2015): e0124708
WGA-Alexa transsynaptic labeling in the phrenic motor system of adult rats: Intrapleural injection versus intradiaphragmatic injection.
Authors: Buttry, Janelle L and Goshgarian, Harry G
Journal: Journal of neuroscience methods (2015): 137-45
Total chemical synthesis of biologically active fluorescent dye-labeled Ts1 toxin.
Authors: Dang, Bobo and Kubota, Tomoya and Correa, Ana M and Bezanilla, Francisco and Kent, Stephen B H
Journal: Angewandte Chemie (International ed. in English) (2014): 8970-4
Label-free real-time acoustic sensing of microvesicle release from prostate cancer (PC3) cells using a Quartz Crystal Microbalance.
Authors: Stratton, Dan and Lange, Sigrun and Kholia, Sharad and Jorfi, Samireh and Antwi-Baffour, Samuel and Inal, Jameel
Journal: Biochemical and biophysical research communications (2014): 619-24
Illuminating the off-pathway nature of the molten globule folding intermediate of an α-β parallel protein.
Authors: Lindhoud, Simon and Westphal, Adrie H and Borst, Jan Willem and van Mierlo, Carlo P M
Journal: PloS one (2012): e45746