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ReadiUse™ Preactivated APC Maleimide [Activated Allophycocyanin]

Flow cytometry analysis of whole blood cells stained with CD8 (SK1)-APC conjugate prepared with ReadiUse™ Preactivated APC Maleimide (Cat No. 2567). The fluorescence signal was monitored using an Aurora flow cytometer in the R1-A channel
Flow cytometry analysis of whole blood cells stained with CD8 (SK1)-APC conjugate prepared with ReadiUse™ Preactivated APC Maleimide (Cat No. 2567). The fluorescence signal was monitored using an Aurora flow cytometer in the R1-A channel
Ordering information
Price ()
Catalog Number2567
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
SolventWater
Spectral properties
Extinction coefficient (cm -1 M -1)700000
Excitation (nm)651
Emission (nm)660
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageRefrigerated (2-8 °C); Minimize light exposure
UNSPSC12171501
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OverviewpdfSDSpdfProtocol


Extinction coefficient (cm -1 M -1)
700000
Excitation (nm)
651
Emission (nm)
660
ReadiUse™ Preactivated APC Maleimide is prepared by reacting highly purified allophycocyanin (APC) with SMCC. The NHS group of SMCC reacts with the lysine groups of APC, leaving maleimide groups available to react with free sulfhydryl groups on proteins to be conjugated. ReadiUse™ Preactivated APC Maleimide is highly purified to completely remove the unreacted SMCC, and lyophilized to powder. It is ready to use and will conjugate without further preparation upon mixing with sulfhydryl-containing target molecules such as reduced IgGs. The activated allophycocyanin can be easily conjugated to antibodies and other proteins without the use of added chemical crosslinking agents. These highly purified phycobiliproteins maintain their spectral characteristics of APC.

Example protocol


SAMPLE EXPERIMENTAL PROTOCOL

Reduction of Antibody
  1. Prepare a fresh solution of 1.0 M DTT (15.4 mg/100 µL) in distilled water. Antibody reduction can be carried out in different buffers, such as MES, phosphate, or TRIS (pH 6 to 8). For best results, antibody solutions should be >2 mg/mL. If less than 2 mg/mL, the antibody should be concentrated.
  2. Add 2 µL of 1.0 M DTT stock per 100 µL of antibody solution, and mix well. Let the antibody solution stand at room temperature for 30 minutes without additional mixing to minimize the reoxidation of cysteines.
  3. Purify the reduced antibody using a desalting column (Cat No. 60500) pre-equilibrated with 50 mM MES buffer (pH 6.0-6.5) and 2 mM EDTA.
  4. Measure the antibody concentration using a Nanodrop. (Con. (mg/mL) = A280nm/1.4).
    Note     Reduced antibodies are typically unstable. It is best to run the conjugation reaction immediately following purification.
     

Conjugate with ReadiUse™ Preactivated APC Maleimide
  1. Reconstitute ReadiUse™ Preactivated APC Maleimide in ddH2O to make a 10 mg/mL solution. For Cat No. 2567 (1 mg) reconstitute in 100 µL of ddH2O. If using Cat No. 2568 (5 mg), reconstitute in 500 µL of ddH2O. Reconstituted ReadiUse™ Preactivated APC Maleimide solutions are stable at 4 °C for up to one month, protected from light.
  2. Add the reduced antibody directly to the solution of ReadiUse™ Preactivated APC at a ratio of 130 µg APC/100 µg of reduced antibody.
  3. Rotate the mixture for 60 to 120 minutes at room temperature.
  4. Block any free sulfhydryl groups on the antibody. Prepare a fresh solution of 10 mg/mL N-Ethylmaleimide (NEM) in DMSO and add 3.4 µL per mg of antibody. Rotate for 20 minutes at room temperature. 

Conjugate Purification
  1. Purify the antibody/APC conjugate solution using size exclusion chromatography.
    Note     Typically the antibody/APC conjugate can be stored at 2-8 °C, protected from light, for up to 6 months. The best storage conditions for any particular conjugate must be determined by experimentation and depends on the stability of the antibody.
     

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Extinction coefficient (cm -1 M -1)700000
Excitation (nm)651
Emission (nm)660

Product family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)
ReadiUse™ Preactivated HRP maleimide---
ReadiUse™ Preactivated PE Maleimide [Activated R-Phycoerythrin]5665741960000
ReadiUse™ Preactivated PerCP Maleimide477678350000

References


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