A Brief Look on Protein Sequencing
Tags: protein sequencing
We talk about the protein sequencing is mainly about the primari structure of protein sequencing. This primary structure includes the the number of polypeptide chains in protein. And in many cases, polypeptides and protein can be used as the one. The oder of the acid amino in polypeptide is the bio-functional bases of protein.
Requirment of the sequencing
Sample purity(over 97%)
Accurate molecular weight of proteins
Accurate number of subunits
Determination of amino acid composition of the protein; and calculate the number of each amino acid in the molecular weight.
Determination of the amount of ammonia in the hydrolysis, calculated amide content
Determination of protein and amino acid sequence of a polypeptide
Open and isolation of peptide chain
The number determination of protein molecules in the polypeptide chain
Break of disulfide bonds
Determination of the amino acid composition of each polypeptide chain, and calculate the molecular ratio of amino acid components
Determination of the N-terminal, C terminal
Polypeptide chain scission
Determination of the amino acid sequence of each peptide.
The steps of sequencing
- Split the polypeptide chain
Protein molecules are composed of multiple polypeptide chains, it must be firstly splited. Several polypeptide chains are linked together with the help of non-covalently, which is known as oligomeric proteins.
- To determine the number of the polypeptides in protein.
Determination of the number of the polypeptide chain in protein molecules. Through the determination of amino acid residues at the end of the number of moles and the relationship between the protein molecular weight, can determine the number of the polypeptide chain..
- Disulfide bond rupture.
Several polypeptide chain through disulfide bond together, can be in 8 mol/L urea or, in the presence of 6 mol/L guanidine hydrochloride with excessive – mercaptoethanol processing, make the disulfide bond reduction of sulphur, and then use generated thiol alkylating reagent protection, to prevent the oxidation of the it to be.
- To determine the amino acid composition, each of the polypeptide chain and calculate the amino acid composition of molecular ratio
- Analyzing the N – end of a polypeptide chain and C – end
- Polypeptide chain rupture into multiple peptides. Two or more different fracture method can be used to peptide broken into two or more sets of peptides or peptide fragments, and the separation
- The determination of each amino acid sequence of peptides
- Determine the peptides sequence in the polypeptide chain. To use two or more sets of amino acid sequence of peptides crisscross overlap each other, piecing together the whole amino acid sequence of a polypeptide chain.
- To determine the location of disulfide bond in the polypeptide chain
Those are the steps of protein sequencing. And you can take those tips to achieve your goals.