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Reduction and s-carboxymethylation of proteins: microscale method
CSH Protocols
|March 2, 2011
Summary
This study presents a new protocol for reducing and alkylating microscale protein samples. The method minimizes sample loss, achieving over 90% yield for protein reduction and S-carboxymethylation.
Area of Science:
- Biochemistry
- Proteomics
Background:
- Traditional protein reduction and alkylation methods cause significant sample loss with microscale protein amounts (10-50 μg).
- Desalting and dialysis are unsuitable for low-microgram protein quantities due to substantial sample loss.
Purpose of the Study:
- To describe an optimized protocol for the reduction and S-carboxymethylation of low-microgram protein samples.
- To minimize sample loss during protein modification procedures.
Main Methods:
- Protein samples (10-50 μg) are reduced and alkylated in small volumes (<200 μl) with detergent.
- Reverse-phase high-performance liquid chromatography (RP-HPLC) is used for desalting and concentrating modified proteins.
Main Results:
- The described protocol yields over 90% for protein reduction and S-carboxymethylation.
- Detergent inclusion during manipulation further reduces protein loss.
Conclusions:
- This protocol offers an efficient method for modifying low-microgram protein amounts.
- The procedure is suitable for applications requiring high recovery of modified proteins.
