Experiments are the most important for scientists. However, the success of the experiment is affected by many factors. Then how can we avoid mistakes during experiments and achieve our aims are the biggest question. Here I will introduce the common mistakes during the protein array sequencing. If you are facing with these problems, you are lucky now.
The process of protein array sequencing
The first step is Splitting the polypeptide chain
The protein molecules, which are composed of multiple polypeptide chains, should be split at the first step. Oligomeric proteins is formed by the several polypeptide chains linked by non-covalently. For instance, hemoglobin is tetramer, dimer is enolase; we can use 8mol / L urea or 6mol / L guanidine hydrochloride to separate polypeptide chains (subunits). The second step is to determine the number of polypeptide chain in protein molecules. You can determine the number of polypeptide chains by measuring the relationship between the number of terminal amino acid residues and protein molecular weight. Next is disulfide bond breakage. When several polypeptide chains cross-linked by disulfide, you can firstly use an excess of mercaptoethanol to make disulfide bonds be into the mercapto under the circumstance of 8mol / L urea or 6mol / L guanidine hydrochloride. Then you can determine the amino acid in multiple polypeptide chains to calculate the molecular ratio of amino acid components. And analyzing the N-and C-terminal polypeptide chain are very important. Polypeptides will break into a plurality of peptides. Then you can determine the order of the amino acid in peptides and the order of peptide in polypeptide chain. The last one is to determine the original location of disulfide bonds in polypeptide chain.
Equipment of this experiment
The samples’ pure should be over 97%; knowing the molecular weight of the protein; knowing the number of sub-units forming the protein; determine the protein’s composition of amino acid and calculating the number of each amino acid according to the molecular weight; determining the amount of ammonia in the hydrolysis and calculating the content of amide.
Mistakes in this experiment
The first one is the purity of the samples. If samples’ purity is under the 97%, this will affect the result of experiment.
The reason why we sometimes make this mistakes is that we can not find the samples purity enough or the samples before the experiment are polluted by other substances.
During the process of disulfide bonds break, some scientists always forget to use alkylating agent to protect the sulfhydryl in case of re-oxidation. And then the following steps can not be in order. The first one is to be processed in performic acid, then it can be reduced and oxidated.
These two steps can not be changed.
This kinds of mistakes occur because we are not careful enough. This is the main steps and some mistakes may occur in this kind of experiments.