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AAT Bioquest

Amplite® Fluorimetric Maleimide Quantitation Kit *Green Fluorescence*

N-ethylmaleimide dose response was measured in a 96-well solid black
plate with Amplite™ Fluorimetric Maleimide Quantitation Assay Kit using a
NOVOstar microplate reader (BMG Labtech).
N-ethylmaleimide dose response was measured in a 96-well solid black
plate with Amplite™ Fluorimetric Maleimide Quantitation Assay Kit using a
NOVOstar microplate reader (BMG Labtech).
N-ethylmaleimide dose response was measured in a 96-well solid black
plate with Amplite™ Fluorimetric Maleimide Quantitation Assay Kit using a
NOVOstar microplate reader (BMG Labtech).
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Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


A variety of crosslinking reagents with a maleimide group are widely used for crosslinking proteins to proteins or proteins to other biomolecules. There are few reagents or assay kits available for quantitate the number of maleimide groups that are introduced into the first protein. All the commercial kits have tedious protocols. Our kit uses a proprietary dye that has enhanced fluorescence upon reacting with a maleimide. The kit provides a sensitive, one-step fluorimetric method to detect as little as 10 picomole of maleimide in a 100 µL assay volume (100 nM in concentration). The assay is rapid and robust. It can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation. The kit provides a convenient protocol with all the essential reagents. For the rapid quantification of a protein maleimide group we recommend you use #5526. For the quantification of a nano particle maleimide group we recommend you use #5525.

Platform


Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateSolid black

Components


Example protocol


AT A GLANCE

Protocol Summary
  1. Prepare 20X Maleimide reaction mixture (260 µL)
  2. Incubate at room temperature for 30 - 60 minutes
  3. Prepare N-ethylmaleimide standards or test samples (50 µL)
  4. Add Maleimide working solution (50 µL)
  5. Incubate at RT for 5 to 30 minutes
  6. Monitor the fluorescence increase at Ex/Em = 490/525 nm (Cutoff = 515 nm)
Important Note

Thaw all the kit components at room temperature before starting the experiment.

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

Maleimide GreenTM stock solution (500X)

Add 20 µL of DMSO (Component E) into the vial of Maleimide GreenTM (Component A) to make 500X Maleimide GreenTM stock solution. Note: 10 µL of 500X Maleimide GreenTM stock solution is enough for one 96-well plate.

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/5523

N-ethylaleimide standard
Add 10 μL of 10 mM (10 nmol/µL) N-ethylmaleimide Standard (Component D) to 990 μL of Assay Buffer (Component C) to generate 100 μM (100 pmol/µL) N-ethylmaleimide standard solution. Take 100 μM (100 pmol/µL) N-ethylaleimide standard solution and dilute to 10 μM. From 10 μM (MS7) standard solution, perform 1:2 serial dilutions to get serially diluted N-ethylmaleimide standards (MS6-MS1) with Assay Buffer (Component C).

PREPARATION OF WORKING SOLUTION

Add 10 μL of 500X Maleimide GreenTM stock solution into 250 μL Reaction Buffer (Component B) and mix well to make 20X Maleimide reaction mixture. Incubate 20X Maleimide reaction mixture at room temperature for at least 30 minutes, protected from light. Note: It is very important to incubate the 20X Maleimide reaction mixture for at least 30 mins to maximize the signal to background ratio. Note: You should see the yellow color after adding the 500X Maleimide GreenTM stock solution into Reaction Buffer (Component B).

Add the whole bottle of 20X Maleimide reaction mixture into 5 mL of Assay Buffer (Component C) and mix well to make Maleimide working solution. Note: This Maleimide working solution is not stable. Use within 1 hour.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of N-ethylmaleimide standards and test samples in a solid black 96-well microplate. MS = N-ethylmaleimide Standards (MS1 - MS7, 0.14 to 10 µM); BL=Blank Control; TS=Test Samples

BLBLTSTS
MS1MS1......
MS2MS2......
MS3MS3
MS4MS4
MS5MS5
MS6MS6
MS7MS7

Table 2. Reagent composition for each well.

WellVolumeReagent
SD1-SD750 µL

Serial Dilutions (0.14 to 10 µM)

BL50 µLAssay Buffer
TS50 µLTest Sample
  1. Prepare N-ethylmaleimide standards (MS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
  2. Add 50 µL of Maleimide working solution to each well of N-ethylmaleimide standard, blank control and test samples to make the total Maleimide assay volume 100 µL/well. For a 384-well plate, add 25 µL of Maleimide working solution into each well instead, for total volume of 50 µL/well.
  3. Incubate the reaction at room temperature for 5 to 30 minutes, protected from light. Note: For best results, the fluorescence intenisty should be read within 30 minutes due to the fact that the fluorescence background increases with time.

  4. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 490/525 nm (Cutoff = 515 nm).

Images


Citations


View all 10 citations: Citation Explorer
Regulation of pancreatic cancer microenvironment by an intelligent gemcitabine@ nanogel system via in vitro 3D model for promoting therapeutic efficiency
Authors: Chen, Di and Zhu, Xiaofei and Tao, Wanru and Kong, Yan and Huag, Yong and Zhang, Yajun and Liu, Ri and Jiang, Lingong and Tang, Ying and Yu, Haiyan and others,
Journal: Journal of Controlled Release (2020)
Prote{\'\i}nas fluorescentes como sensibilizadores de emiss{\~a}o em nanopart{\'\i}culas contendo Eu3+
Authors: Maturi, Fernando Eduardo
Journal: (2018)
Restricting the conformational freedom of the neuronal nitric-oxide synthase flavoprotein domain reveals impact on electron transfer and catalysis
Authors: Dai, Yue and Haque, Mohammad Mahfuzul and Stuehr, Dennis J
Journal: Journal of Biological Chemistry (2017): 6753--6764
Study of Electron Transfer through the Reductase Domain of Neuronal Nitric Oxide Synthase and Development of Bacterial Nitric Oxide Synthase Inhibitors
Authors: Dai, Yue
Journal: (2016)
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


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