c***y
发帖数: 233 | 1 请问那里可以找到一般的protocol?
反应是怎么进行的呢？Dye的什么基团和amine反应？ | v**********m
发帖数: 5516 | 2 Thermo Fisher Scientific
【在 c***y 的大作中提到】
: 请问那里可以找到一般的protocol?
: 反应是怎么进行的呢？Dye的什么基团和amine反应？
| b******y
发帖数: 627 | | s****n
发帖数: 234 | 4 This is how we make home-made Halo-Atto647N in our lab using amine/NHS ester
chemistry. It is probably not the most cost-efficient way, but we did get
huge amount of Halo-Atto647N. Hope this would help.
Reagents and Reaction Set Up:
Dissolve 5mg of Atto NHS ester in 400ul of Anhydrous N,N DMF. (Final
Concentration 10mM)
Add 120 ul of 100mM stock solution of Amino HaloTag Ligand. (Final
Concentration 20mM)
Add 3.1 ul of DIPEA (reagent grade) to the solution. (Final Concentration
30mM)
Bring final volumn of reaction to 600ul.
Ratio of Atto NHS ester : Amino HaloTag Ligand : DIPEA is 1:2:3.
Incubate reaction mixture in a glass tube covered in foil at 30C overnight
while tube is rotating.
Note: Atto NHS ester should be between 5-20 mM during reaction. Fewer
equivalents of amino reactant are required because reaction is performed in
organic solvent so NHS ester hydrolysis is less of a concern. Save roughly 2
-3ul of stock solutions of reactants for LCMS analysis after reaction.
Purification of Product:
Use a LCMS (quantities less than 10mg) or HPLC system (quantities greater
than 10mg) to purify the product from the mixture. Reverse phase
chromatography with a C18 column is recommended with an ethanol/methanol:
water gradient and the sample of reactants can be run on the LCMS to assess
the degree of the reaction.
Note: LCMS and HPLC both use the same type of column; the difference between
the two systems is that LCMS runs each sample through the mass spectrometer
to determine where the product is and collects at the appropriate time
while the HPLC uses UV which is a less sensitive method.
Note: NEB, Promega, and Atto company reaction conditions are summarized and
combined here but optimization for specific tag combination may require
adjustment of the conditions.
【在 c***y 的大作中提到】
: 请问那里可以找到一般的protocol?
: 反应是怎么进行的呢？Dye的什么基团和amine反应？
| C*******e
发帖数: 4348 | 5 PIERCE有一整套可以跟primary amine反应label蛋白的
还可以看他们的protein crosslinking书
网上免费下
【在 c***y 的大作中提到】
: 请问那里可以找到一般的protocol?
: 反应是怎么进行的呢？Dye的什么基团和amine反应？
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