There has been carrying out the research to microquantitative determination and recoverying by precipitation of the recombinant protein in solution which includes very little protein by using nigrosin which is produced at Biochemistry Department of Basic Medical Faculty, Pyongyang Medical College of
Microquantitative determination of the recombinant protein has been carried out using only Coomassie brilliant blue G-250 which was developed as protein-binding dye in 1976.
Microquantitative determination of protein is required in several sections of medicine and biology including clinical biochemical diagnosis as well as preparation of the recombinant protein.
Quantitative method of protein using dye-binding method does not needequipments such as centrifuge, electrophoresis apparatus, ultraviolet spectrophotometer and can determine the concentration of protein in solution with the simple procedure for a short time.
In our country, there has been carried out the studies to prepare several bioactive proteins by means of genetic engineering and some recombinant proteins such as human growth hormone, human interferon α2b are used as drugs. Furthermore, it has been tried to prepare other recombinant human proteins including erythropoietin, thymosin and albumin-growth hormone fusion protein.
It is an important issue to determine proteins of solution with low concentration of protein and to analyze by electrophoresis using microsample in every step of preparation process.
Now in our country, microquantification of the recombinant is being carriedout by using Coomassiee brilliant blue(CBB) G-250, which is expensive andpurchased from other countries and method for recovering the microproteinfrom solution has not been developed.
We were going to establish method to directly determine and precipitate very little protein of solution by using nigrosin, which is produced in ourcountry, in stead of Coomassiee brilliant blue G-250.
Optical density of reactive solution was high mostly in 577nm when 0.1mLof 2mg/mL recombinant human growth hormone added into 1mL of 0.01% nigrosin solution.
Optical density of reaction using Coomassiee brilliant blue G-250 was high mostly in 595nm.
Proteins of solutions with concentration ranging 3~5 000㎍/mL could be determined by mixing nigrosin and sample which allowed to contain 0.01% nigrosin in 2% trichloroacetic acid and measuring OD577 of mixture after 4 minutes.
pH suitable to determine protein using nigrosin was in range of 3-9 and difference of optical desity of protein depending on the sort of protein could be overcome by addition of SDS in 0.03%.
Method using nigrosin had the same reliability as that of CBB G-250(r=0.999) and had high reappearance.( Co-appearance; CV<4.6％, Day-appearance; CV<5.1％)
When 1mL of 0.03％ nigrosin solution was added in 1mL of 10~100㎍/mL rhGH and Pellets of all solutions was applied in 10% SDS-PAGE after centrifugation (10 000rpm, 10min), pellets of all solutions were found to have proteins. In solutions before precipitation with nigrosin, samples with concentration of more than 20㎍/mL were detected to have protein after electrophoresis.
We determined the minimal protein concentration of solution in which proteincould be recovered by precipitation with nigrosin.
Protein from solutions with more than 3㎍/mL concentration of protein could be recovered by addition of nigrosin into the solution.
In the future, we are going to introduce this method to the research institution in which bioactive proteins are prepared bionically and the clinical biochemical diagnosis including microquantitative determination of the protein in urine.
