RU2298939C2 - Method for protein isolation from marrow cells - Google Patents

Abstract

FIELD: biochemistry, in particular protein isolation from marrow cells of animal tubular bones.

SUBSTANCE: claimed method includes marrow cell isolation, suspending thereof, suspension filtration, filtrate centrifugation. Further precipitates together with supernatants in centrifugal test-tubes are triply frozen and thawed in refrigerated room, filtered through nylon-6 filter and precipitated with 10 % trichloroacetic acid solution at 60°C, settled for 12 h at 4°C and centrifuged at 1700 rpm for 5 min. Protein solution is further subjected to dialysis with flowing water for 1-2 days and distilled water for 1 hour.

EFFECT: simplified method; target product of improved quality.

3 ex

Description

The invention relates to biochemistry, namely, the allocation of proteins from bone marrow cells of the bones of animals.

A known method of protein synthesis described in RU 2041716 C1, A61K 35/28, 08/20/1995.

The disadvantages of this method are the complexity, duration in time and compliance with special conditions of protein synthesis. In addition, the final product is not clean enough.

A simplification of the method for isolating total protein from the tubular bones of the bone marrow and increasing the purity of the target product is achieved by the fact that bone marrow cells are taken from agricultural and wild animals. The tubular bones are separated from the flesh. In laboratory animals and birds, holes are created in the upper and lower parts of the bone; in large animals, bone is cut. Femoral bone marrow cells are suspended by flushing through the created openings with a 199 medium syringe into the flask. Thoroughly mix the contents of the flask to a uniform composition by pipetting. The resulting cell suspensions are passed through nylon filters d 10 cm for thorough purification from the yellow marrow and washed 3 times with chilled medium 199 (or Needle). Filters are prepared from nylon products of the textile industry. Before use, the filters are boiled in distilled water 3 times for 15 minutes. The filtrate is subjected to centrifugation for 5-7 minutes at 3 thousand rpm Precipitation together with supernatants in centrifuge tubes is subjected to three freezing-thawing in a refrigerator, which results in the destruction of the cell walls of the sediment from the bone marrow (which is then discarded) and additional protein molecules are released into the supernatant, which affects the yield of the final product. To isolate the protein and release it from debris, the contents of the centrifuge tubes are filtered through nylon filters. Smears are made from the precipitate, stained according to generally accepted methods and examined under a microscope - the cells are not detected in them (homogeneous mass), therefore, all biologically active substances pass into solution.

The selection of total protein is as follows.

In glasses for centrifugation with a protein solution add a 10% solution of trichloroacetic acid based on 5 drops of a protein solution, 2 drops of an acid solution. For better protein precipitation, the contents of the glasses are heated to a temperature of 40-60 ° C (in the case that the proteins precipitate poorly). The extracts for complete precipitation leave for 12 hours in a refrigerator at a temperature of + 4 ° C. Protein precipitate is separated from the solution by centrifugation at 1700 rpm for 5 min — all of the above methods, their exposures and modes were established experimentally, due to which this method and the final result are carried out most fully, i.e. more complete deposition and separation of bone marrow protein from the solution. Precipitation is washed off with a small amount of alcohol, then ether and transferred to a watch glass for drying. The proteins thus obtained for further purification are subjected to dialysis in order to completely remove the acid and restore the hydration shells of the peptide. For this, the protein solution is placed in a container separated from the low molecular weight solution (flowing or distilled water) by a cellophane membrane. Dialysis is carried out with running water for 1-2 days with continuous supply and distilled water for 1-2 hours. The absence of acid is controlled by a neutral reaction with an indicator.

Example 1

The femurs from 5 piglets are separated from the flesh, then the bones are cut from two sides in the center and the bone contents are washed with 199 medium into the flask. Cell suspensions are filtered through nylon filters d 10 cm, which are pre-sterilized. The filtrate is centrifuged for 7 minutes at 3 thousand rpm. The precipitate and supernatant are subjected to three freezing-thawing. In the process, cells of the suspended sediment by medium 199 are counted at a dilution of 1:10 in the Goryaev chamber. At the last thawing, a homogeneous mass is detected in the sediment. The contents of the centrifuge tubes are again filtered through nylon filters. Proteins are precipitated from a solution of 10% trichloroacetic acid based on 5 drops of a protein solution, 2 drops of trichloroacetic acid are left to stand for 12 hours. The precipitate is separated from the solution by centrifugation at 1700 rpm for 5 minutes. Precipitation is washed off with alcohol and ether, dried on a watch glass, and weighed.

The weight of the total protein isolated from the bone marrow of piglets is 200 ± 0.26 mg. Protein is a dry amorphous powder of gray-brown color.

Example 2

Tubular bones from 3 puppies of foxes are separated from the flesh. Preparation of bone contents for protein isolation is carried out as described in the previous example. Proteins from the solution are precipitated with trichloroacetic acid (10% solution) based on 5 drops of a protein solution, 2 drops of trichloroacetic acid.

The contents of the flask are heated to a temperature of about 60 ° C, left for 12 hours in the refrigerator at a temperature of + 4 ° C. The next day, abundant flakes of precipitated protein are observed in the flask. The precipitate is treated and dried as described above. The weight of the total protein from the brain substance of fox puppies isolated by this method is 136.6 ± 8.9 mg. Protein is an amorphous brown-brown powder.

(In examples 1 and 2, as in example 3, the technical operations for obtaining bone marrow proteins are the same and the dialysis process in them is also carried out for additional purification during their further use, however, how dialysis is carried out is described only in the third example, so as not to repeat and diversify the example).

Example 3

Tubular bones from 3 sables are separated from the flesh. The leaching of cell populations, the elimination of fatty layers and the isolation of protein are carried out as described in the previous examples. The weight of total sable bone marrow protein is 50.0 ± 2.8 mg. The dry crystalline sable bone marrow protein is transferred to the solution. To do this, take 0.05 g of protein and add 0.45 ml of 0.9% sodium chloride solution, mix thoroughly. The protein solution is dialyzed by placing it in a container with a cellophane membrane. This container is placed in a bowl with running water, which is supplied continuously through a hose. Dialysis in running water takes 24 hours (pH 3.5-4.0). Then, in distilled water, dialysis proceeds for 1 h (neutral indicator response). The total protein content is determined using a refractometer type IRF-22 according to the standard method. The amount of total protein in sable's bone marrow is 6.77%.

A distinctive advantage of the proposed method for the isolation of proteins from the tubular bones of the bone marrow is accessibility, starting from the use of nylon filters, reagents and including the ease of technical operations, which allows more efficient filtration and simplifies the method.

Claims (1)

A method for isolating proteins from bone marrow cells, which involves removing bone marrow cells, suspending them, filtering the suspension, centrifuging the filtrate, characterized in that after centrifugation of the precipitate, the supernatants in centrifuge tubes are subjected to three freezing-thawing in a refrigerator, then the contents of the centrifuge the tubes are filtered through nylon filters, the proteins from the solution are precipitated with a 10% trichloroacetic acid solution when heated to 60 ° C, standing by 12 hours at 4 ° C and centrifuged at 1700 rev / min for 5 minutes, then the protein solution was dialysed in running water for 1-2 days, and distilled water for 1 hour.