Which amino acid would Iodoacetate react with?
Iodoacetate Derivatives Iodoacetate (and bromoacetate) can react with several nucleophilic functional groups within proteins. Their relative reactivity toward protein functionalities is sulfhydryl>imidazolyl>thioether>amine.
Is TCEP a reducing agent?
Thermo Scientific Pierce TCEP-HCl is a potent, versatile, odorless, thiol-free reducing agent with broad application to protein and other research involving reduction of disulfide bonds.
What does Iodoacetate do to proteins?
Iodoacetate derivatives have been used for decades to block or crosslink sulfhydryl groups in proteins and other molecules (Chapter 2, Section 5.2). At mildly alkaline pH values (pH 8–8.5), iodoacetyl derivatives are almost entirely selective toward the cysteine SH groups in proteins.
What kind of inhibitor is Iodoacetate?
Peptidase inhibitor Iodoacetate is an irreversible inhibitor of all cysteine peptidases, with the mechanism of inhibition occurring from alkylation of the catalytic cysteine residue (see schematic). In comparison with its amide derivative, iodoacetamide, iodoacetate reacts substantially slower.
What is the difference between TCEP and DTT?
For long-term storage of proteins, TCEP is significantly more stable than DTT without metal chelates such as EGTA in the buffer, whereas DTT is more stable if metal chelates are present. Thus TCEP has advantages over DTT, although the choice of reductant is application specific.
What is the reaction rate of maleimide with amines?
At pH 7.0, the reaction rate of maleimide with thiols is about 1,000 times faster than the reaction rate of maleimide with amines. However, above pH 7.5, free primary amines react competitively with thiols at the maleimide C=C bond (Figure 3).1 Figure 4: The thiol-maleimide reaction. (a) The reaction is chemoselective for thiols from pH 6.5 – 7.5.
What is a maleimide thiol reaction?
Read more in our related article. The maleimide thiol reaction mechanism involves a Michael addition reaction where the maleimide acts as the Michael acceptor while the thiolate is the Michael donor. Depending on how strong a base the nucleophile is, it will undergo either direct addition or conjugate addition.
How do maleimides react with cysteine?
Maleimide-based labels and cross-linkers react with cysteine to form a thioester bond, which is reported to be resistant to reversal by reducing agents (Fig. 2 C). This oft-repeated boon of maleimide chemistry is one of the main advantages maleimides have over MTS reagents, especially if subsequent reactions utilize reducing agents.
Why are maleimides used in conjugation chemistry?
Maleimides represent one of the most widely used functional groups for conjugation chemistry. 1–4 This is due to the extremely favourable kinetics of their reaction with thiols, which ensures high yields of the desired conjugates are generated rapidly whilst minimising competing side-reactions.