Is maleimide a fluorescent?
N-(1-Pyrene)maleimide is nonfluorescent in aqueous solution but forms strongly fluorescent adducts with sulfhydryl groups of organic compounds or proteins.
How do you neutralize TCEP?
(Note: TCEP solutions are commonly neutralized due to having a pH of ~2.5 in water.) Add 5.733 g of TCEP to a 50 ml conical vial, making sure to get as much of the TCEP as possible into the vial. Add 35 ml of cold molecular biology grade water to the vial, and dissolve the TCEP.
What is SH group called?
The ―SH group of a thiol is known as a mercapto group, and therefore the prefix mercapto- may be included in the names of certain compounds—for example, 2-mercaptoethanol or 2-mercaptobenzothiazole. When a thiol is named relative to a corresponding oxygen compound, the prefix thiol- is used—for example, thiophenol.
Is TCEP reversible?
TCEP reaction is irreversible, whereas DTT reaction is reversible.
Why is TCEP used?
TCEP is often used as a reducing agent to break disulfide bonds within and between proteins as a preparatory step for gel electrophoresis.
Why is sulfur a good leaving group?
The positive charge on the sulfur also makes it an excellent leaving group, as the resulting product will be a neutral and very stable sulfide. All in all, in both reactions we have a reasonably good nucleophile, an electron-poor, unhindered electrophile, and an excellent leaving group.
What is SH functional group?
Definition. In organic chemistry, a thiol is a compound that contains the –SH functional group, which is the sulfur analog of a hydroxyl or alcohol group. The functional group is referred to as either a thiol group or a sulfhydryl group. Thiols are more traditionally referred to as mercaptans.
What is maleimide reaction in chemistry?
Maleimide reaction chemistry. Maleimide reaction scheme for chemical conjugation to a sulfhydryl. R represents a labeling reagent or one end of a crosslinker having the maleimide reactive group; P represents a protein or other molecule that contains the target functional group (i.e., sulfhydryl, –SH).
Can Peg-maleimide be conjugated with TCEP as reducing agent?
I’ve been tried conjugating proteins with PEG-maleimide using TCEP as reducing agent and it completely failed. Indeed, I found in the bibliography that PEG interferes somehow with TCEP. Could be a matter of TCEP concentration? Which should be the optimal ratio protein:TCEP:PEG-maleimide? Thank you!
What is the best way to reduce and label CF® dye maleimide?
The reduction reaction and the subsequent labeling reaction are best carried out in the presence of an inert gas (N2 or Ar) to prevent re-formation of disulfide bonds. Allow the vial of CF® dye maleimide to warm up to room temperature. Prepare a 10 mM dye stock solution. For 1 umol dye: add 100 uL anhydrous DMSO to the vial.
What is the solubility of maleimides?
Maleimides with good aqueous solubility (like sulfo-Cy maleimides) can be dissolved in water. If precipitation occurs, increase content of organic co-solvent in the mixture to achieve better labeling. Dialysis is recommended as a means of purification only for maleimides with good aqueous solubility.