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Amplite® Colorimetric Maleimide Quantitation Kit

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Telephone1-800-990-8053
Fax1-800-609-2943
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Maleimides can be directly assayed spectrophotometrically at 302 nm. However, the small extinction coefficient of 620 M-1cm-1 renders this assay insensitive, and the assay is further complicated by the protein absorbance at the same wavelength. This colorimetric maleimide assay kit quantifies maleimide groups by first reacting a sample with a known amount of thiol present in excess and then assaying the remaining unreacted thiol using 4,4\'-DTDP with a molar extinction coefficient of 19,800 M -1cm-1. The amount of maleimide is calculated as the difference between the initial amount of thiol and the amount of unreacted thiol after the complete reaction of all maleimide groups. This spectrophotometric assay for the determination of maleimide groups is a reverse GSH assay. It takes advantage of the high reactivity of thiols of GSH with the maleimide moiety. Maleimide of the sample is allowed to form a stable thiosuccinimidyl linkage with GSH. After the reaction of the sample is complete, the excess GSH, i.e., the remaining thiols of GSH in the reaction mixture, is estimated by using 4,4\'-DTDP. The amount of GSH reacted with the sample is titrated to determine the extent of maleimide. For more sensitive maleimide quantitation, we recommend that you use our fluorimetric kit # 5523 that has higher sensitivity.

Platform


Absorbance microplate reader

Absorbance324 nm
Recommended plateClear bottom

Components


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. MEA stock solution (500X)
Add 200 µL of distilled water into the vial of MEA (Component A). Note: 10 µL of the 500X MEA stock solution is enough for 50 reactions (0.5 mL/reaction). The unused 500X MEA stock solution should be divided into single use aliquots, stored at -20 oC and kept from light.

2. 4,4'-DTDP stock solution (50X)
Add 1 mL of DMSO (Component D) into the vial of 4,4'-DTDP (Component B), and mix well. Note: 100 µL of the 50X 4,4’-DTDP stock solution is enough for 10 reactions (0.5 mL/reaction). The unused 50X 4,4’-DTDP stock solution should be divided into single use aliquots, stored at -20 oC and kept from light.

PREPARATION OF WORKING SOLUTION

MEA working solution
Add 10 µL of MEA stock solution (500X) into 5 mL of distilled water, and mix them well. Note: The MEA working solution is not stable. We recommend to prepare fresh before use.

SAMPLE EXPERIMENTAL PROTOCOL

The following recommended protocol is for Cuvette.
  1. Set up 3 Total SH tubes: Add 400µL of Assay Buffer (Component C) and 100 µL of MEA working solution into each tube and incubate at room temperature for 20 minutes.
  2. Set up 3 test tubes for each sample: Add 0.05 mg of test sample and sufficient Assay Buffer (Component C) to make the total volume of 400 µL/tube. Add 100 µLof MEA working solution into each tube and incubate at room temperature for 20 minutes..
  3. Measure the absorbance of the Assay Buffer (Component C) as the blank control at 324 nm.
  4. Proceed to Total SH determination while tubes are still incubating (from step 1). Add 10µL of 50X 4,4’-DTDP stock solution into each total SH tube and incubate at room temperature for 2 min. Note: Do not add 50X 4,4’-DTDP stock solution to the sample containing tubes yet.
  5. Measure the absorbance of the 3 Total SH tubes at 324 nm without washing the cuvette. Record the readings and average them to have "ODTSH"
  6. Clean the cuvette and read the absorbance of the first sample tube (from Step 2) at 324 nm (OD0) before add any 4,4’-DTDP stock solution (50X).
  7. Add 10 µL of 4,4’-DTDP stock solution (50X) into the sample cuvette (from Step 6) and mix well. Incubate the sample at room temperature for 2 minutes and read the absorbance at 324 nm (OD). Clean the cuvette, and repeat Steps 6 and 7 for the remaining tubes. Record all readings. 

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Citations


View all 8 citations: Citation Explorer
Perlecan-targeted nanoparticles for drug delivery to triple-negative breast cancer
Authors: Khanna, Vidhi and Kalscheuer, Stephen and Kirtane, Ameya and Zhang, Wenqui and Panyam, Jayanth
Journal: Future Drug Discovery (2019)
Conjugation with eight-arm PEG markedly improves the in vitro activity and prolongs the blood circulation of staphylokinase
Authors: Qi, Fangbing and Hu, Chunyang and Yu, Weili and Hu, Tao
Journal: Bioconjugate chemistry (2018)
Successful acquisition of a neutralizing monoclonal antibody against a novel neutrophil-activating peptide, mitocryptide-1
Authors: Hattori, Tatsuya and Nakashima, Kenta and Marutani, Takayuki and Kiso, Yoshiaki and Nishi, Yoshisuke and Mukai, Hidehito
Journal: Biochemical and biophysical research communications (2015): 54--59
Rapid quantification of maleimide in bioconjuated samples using a novel colorimetric method (TECH2P. 761)
Authors: Liao, Jinfang and Guo, Haitao and Luo, Zhen and Zhao, Qin and Diwu, Jack
Journal: The Journal of Immunology (2015): 206--7
Moderate PEGylation of the carrier protein improves the polysaccharide-specific immunogenicity of meningococcal group A polysaccharide conjugate vaccine
Authors: Zhang, Tingting and Yu, Weili and Wang, Yanfei and Hu, Tao
Journal: Vaccine (2015): 3208--3214
PEG as a spacer arm markedly increases the immunogenicity of meningococcal group Y polysaccharide conjugate vaccine
Authors: Huang, Qingrui and Li, Dongxia and Kang, Aijun and An, Wenqi and Fan, Bei and Ma, Xiaowei and Ma, Guanghui and Su, Zhiguo and Hu, Tao
Journal: Journal of Controlled Release (2013): 382--389
CD133-targeted paclitaxel delivery inhibits local tumor recurrence in a mouse model of breast cancer
Authors: Swaminathan, Suresh Kumar and Roger, Emilie and Toti, Udaya and Niu, Lin and Ohlfest, John R and Panyam, Jayanth
Journal: Journal of Controlled Release (2013): 280--287

References


View all 38 references: Citation Explorer
Characterization of site-specific ScFv PEGylation for tumor-targeting pharmaceuticals
Authors: Natarajan A, Xiong CY, Albrecht H, DeNardo GL, DeNardo SJ.
Journal: Bioconjug Chem (2005): 113
A new maleimide-bound acid-cleavable solid-support reagent for profiling phosphorylation
Authors: Chowdhury SM, Munske GR, Siems WF, Bruce JE.
Journal: Rapid Commun Mass Spectrom (2005): 899
Quantitation of microparticles released from coated-platelets
Authors: Dale GL, Remenyi G, Friese P.
Journal: J Thromb Haemost (2005): 2081
Determination of WR-1065 in human blood by high-performance liquid chromatography following fluorescent derivatization by a maleimide reagent ThioGlo3
Authors: Chen J, Lu Z, Lawrence TS, Smith DE.
Journal: J Chromatogr B Analyt Technol Biomed Life Sci (2005): 161
Reactions of cysteines substituted in the amphipathic N-terminal tail of a bacterial potassium channel with hydrophilic and hydrophobic maleimides
Authors: Li J, Xu Q, Cortes DM, Perozo E, Laskey A, Karlin A.
Journal: Proc Natl Acad Sci U S A (2002): 11605
Resolution, detection, and characterization of redox conformers of human HSF1
Authors: Manalo DJ, Liu AY.
Journal: J Biol Chem (2001): 23554
Site-directed mutation of conserved cysteine residues does not inactivate the Streptococcus pyogenes hyaluronan synthase
Authors: Heldermon CD, Tlapak-Simmons VL, Baggenstoss BA, Weigel PH.
Journal: Glycobiology (2001): 1017
Quantitation of signal transduction
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Journal: Faseb J (2000): 2581
Determination of S-nitrosoglutathione in human and rat plasma by high-performance liquid chromatography with fluorescence and ultraviolet absorbance detection after precolumn derivatization with o-phthalaldehyde
Authors: Tsikas D, S and mann J, Holzberg D, Pantazis P, Raida M, Frolich JC.
Journal: Anal Biochem (1999): 32
Peroxynitrite modification of protein thiols: oxidation, nitrosylation, and S-glutathiolation of functionally important cysteine residue(s) in the sarcoplasmic reticulum Ca-ATPase
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Journal: Biochemistry (1999): 12408