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ReadiUse™ Preactivated APC Maleimide [Activated Allophycocyanin]
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
- 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.
- 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.
- 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.
- 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
- 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.
- Add the reduced antibody directly to the solution of ReadiUse™ Preactivated APC at a ratio of 130 µg APC/100 µg of reduced antibody.
- Rotate the mixture for 60 to 120 minutes at room temperature.
- 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
- 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
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Correction Factor (280 nm) |
| ReadiUse™ Preactivated PE Maleimide [Activated R-Phycoerythrin] | 565 | 574 | 1960000 | 0.175 |
| ReadiUse™ Preactivated PerCP Maleimide | 477 | 678 | 406000 | 0.22 |
References
View all 13 references: Citation Explorer
Magnetic particles encoding a suspension probe for ultra-sensitive and quantitative determination of atrazine.
Authors: Zhao, Zhigao and Dou, Xiaowen and Luo, Jiaoyang and Jin, Meiqi and Qin, Jiaan and Wang, Changjian and Yang, Shihai and Yang, Meihua
Journal: Journal of pharmaceutical and biomedical analysis (2021): 113868
Authors: Zhao, Zhigao and Dou, Xiaowen and Luo, Jiaoyang and Jin, Meiqi and Qin, Jiaan and Wang, Changjian and Yang, Shihai and Yang, Meihua
Journal: Journal of pharmaceutical and biomedical analysis (2021): 113868
Non-oxidative band-3 clustering agents cause the externalization of phosphatidylserine on erythrocyte surfaces by a calcium-independent mechanism.
Authors: Koshkaryev, Alexander and Livshits, Leonid and Pajic-Lijakovic, Ivana and Gural, Alexander and Barshtein, Gregory and Yedgar, Saul
Journal: Biochimica et biophysica acta. Biomembranes (2020): 183231
Authors: Koshkaryev, Alexander and Livshits, Leonid and Pajic-Lijakovic, Ivana and Gural, Alexander and Barshtein, Gregory and Yedgar, Saul
Journal: Biochimica et biophysica acta. Biomembranes (2020): 183231
Leukocyte-Derived Extracellular Vesicles in Blood with and without EpCAM Enrichment.
Authors: Nanou, Afroditi and Zeune, Leonie L and Terstappen, Leon W M M
Journal: Cells (2019)
Authors: Nanou, Afroditi and Zeune, Leonie L and Terstappen, Leon W M M
Journal: Cells (2019)
Exogenous IL-9 Ameliorates Experimental Autoimmune Myasthenia Gravis Symptoms in Rats.
Authors: Yao, Xiuhua and Zhao, Jiarui and Kong, Qingfei and Xie, Xiaoli and Wang, Jinghua and Sun, Bo and Xu, Lixia and Mu, Lili and Li, Hulun
Journal: Immunological investigations (2018): 712-724
Authors: Yao, Xiuhua and Zhao, Jiarui and Kong, Qingfei and Xie, Xiaoli and Wang, Jinghua and Sun, Bo and Xu, Lixia and Mu, Lili and Li, Hulun
Journal: Immunological investigations (2018): 712-724
A high-throughput neutralizing assay for antibodies and sera against hepatitis E virus.
Authors: Cai, Wei and Tang, Zi-Min and Wen, Gui-Ping and Wang, Si-Ling and Ji, Wen-Fang and Yang, Min and Ying, Dong and Zheng, Zi-Zheng and Xia, Ning-Shao
Journal: Scientific reports (2016): 25141
Authors: Cai, Wei and Tang, Zi-Min and Wen, Gui-Ping and Wang, Si-Ling and Ji, Wen-Fang and Yang, Min and Ying, Dong and Zheng, Zi-Zheng and Xia, Ning-Shao
Journal: Scientific reports (2016): 25141
Page updated on November 21, 2024
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