JPS61500810A - Electrophoresis method for separating hemoglobin variants and improved electrophoresis gel used therein - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Electrophoresis technique for separating hemoglobin variants and improved electricity used therein The present invention is an electrophoretic gel for the separation of hemoglobin variants. The present invention relates to pneumophoresis and electrophoresis gels used therein.

2. Description of prior art Electrophoresis method for separating hemoglobin variants and electrophoresis gel used therein is well known to those skilled in the art (1-S). Generally, hemoglobin variant isolation The electrophoresis gel used for this purpose is agar and a buffer solution with a pH of about 6 to about 6.5. The buffer is citrate and ethylenediaminetetraacetic acid ( EDTA).

Problems with prior art electrophoretic methods for separating hemoglobin variants are (a) Formation of chevron-shaped bands of hemoglobin (Hb) A, S, and C; (b) Hbs and low separation of HbS; and (c) the migration distance between HbF and HbA depends on the relative concentration. It's about being dependent.

Therefore, hemoglobin mutations that alleviate or eliminate one or more of the above problems It would be highly desirable to have an electrophoretic method for separating the cells.

Summary of the invention In accordance with the present invention, one or more of the above problems are alleviated or eliminated. Hemogro An electrophoretic method for bin variant separation is provided. The electrophoresis method of the present invention includes (a ) Add the bottom single sample to be tested to the electrophoresis gel; (b) Electrophoresis of step (a) The moving gel is subjected to electrophoresis; (C) The electrophoresed gel of step (b) is fixed; (d ) Drying the fixed electrophoresis gel of step (c). This is the type of method.

A feature of the improved electrophoresis method of the present invention is the use of a novel electrophoresis gel. new Regular electrophoresis gels have a pH of about 5.5 to about 6.5.

(a) a polysaccharide selected from the group consisting of agar, agarose, and mixtures thereof; (b) a buffer; and (c) an alginate and an alginate ester. A composition selected from the group consisting of: Due to the presence of this composition in the electrophoretic gel, At least one of the above problems can be alleviated or eliminated.

Other features and additional advantages of the invention follow a detailed description of preferred experimental embodiments. It will be clear to a person skilled in the art after reading:

Description of preferred embodiments Alginates that can be used in the present invention include lithium, sodium, potassium and and ammonium salts. However, it is not limited to this. alginate Preferably the salt is sodium alginate. alginate - if it contains However, it is also preferable to contain a slight amount of calcilum. alginate s As for polymers, such alginate esters can be used for polypropylene up to 80%. Preferably, it is esterified with gelcol.

A preferred polysaccharide for use in the electrophoretic gels of the present invention is agarose. agaro The base is low electroosmotic agarose.

Either medium electroosmotic agarose or high electroosmotic agarose may be used. The electricity of the present invention It is even more preferable that the polysaccharide used in the pneumophoresis gel is a low electroosmotic agarose. Delicious.

Buffers that can be used in the present invention include adipic acid, citric acid, and ethylenediaminetetraacetic acid. acids, glutaric acid, itaconic acid.

Maleic acid, malic acid, malonic acid, succinamic acid.

Contains succinic acid and tricarballylic acid. However, it is not limited to these .

The electrophoresis gel of the present invention may optionally contain a preservative in addition to these. typical Antibiotics are a preservative.

Includes, but is not limited to, halogenated organic compounds and inorganic compounds. do not have. Readily available preservatives that can be used here include azide), IJum and Ethylmerlithiosalicylic acid, sodium salt.

The electrophoretic gel of the invention optionally comprises an alkali having 2 to 6 carbon atoms and 2 to 4 hydroxyl groups. Kill polyols may also be included. Suitable alkyl polyols that can be used here 2, ethylene glycol, furopanediol, phthanediol, bentanediol Contains ol and glycerol. However, it is not limited to these.

Preferably, the alkyl polyol has 2 to 4 carbon atoms.

The precise concentrations of the various components used in the electrophoretic gels of the invention are not critical.

However, the electroosmotic gel of the present invention has a low electroosmotic aggregation rate of about 0.5 to about 1.2%. loin; about 0.1 to about 1% sodium alginate; an effective amount of preservative; and Contains a quantified amount of alkyl polyol. More preferable is the electrophoresis method of the present invention. low electroosmotic agarose with a gel of about 0.7 to about 1; and about 0.5 to about 0.7 It contains sodium alginate. The nine electrophoresis methods of the present invention are optimal. Low electroosmotic agarose with a dynamic gel of about 0.8%; sodium alginate with a concentration of about 0.6% ; about 0.01% ethyl meleuthiosalicylic acid, sodium salt; 5% 1.2-propylene Ropanediol and 0.2% maleic acid and about 1% disodium maleate. containing about 0.08M buffer consisting of The electrophoretic gels of the present invention can be prepared by any method well known to those skilled in the art.

Generally, a gel solution is prepared by mixing the various components therein, during which time the mixture is approximately 80% It is made by heating to a temperature of ~100°C.

Electrophoretic gels can be made by standard molding or casting methods. That gel is convenient For example, about 2° to about 40°C, preferably about 18° to about 26°C. Preferably, it is stored in a sealed plastic tray.

If the sample is prepared using the method used in the prior art, for example microliquid, then the gel is soaked in alcohol. : Acetic acid mixture 9, e.g. 60% Song alcohol, 30% deionized water, and 10 % glacial acetic acid. Moreover, the gel can be optionally dried at about 80 to about 90°C. Wear.

The following examples are for illustrative purposes only and are intended to illustrate the disclosed invention. It is not a restriction.

Examples 1-6 A gel having the formulation shown in Table 1 was prepared. Each gel contains 0.0581 citrate and 511.2-propanediol (stabilizer) was also used.

The following electrophoretic method was used.

1. Fill each side of the electrophoresis cell with 45 mfi of 0.05 M citrate buffer.

2. Gel - Gently blot the surface of the gel with absorbent paper. Template on the gel surface ). 1. Pipette sample hemolysate 512 with the concentration shown in Table 1. Place it on the clear slot. After placing the last sample, wait 5 minutes and remove excess sample. Remove with absorbent paper on template. Throw away the blotting paper. Remove the template and throw it away. Be careful, two animals In order to infiltrate the gel with a comparable amount of Hb, the agar content was added as the viscosity of the gel increased. The Hb concentration in the gel must be increased.

3. Place the gel on the gel-bridge and place it into the electrophoresis cell. on the bridge at that time Match the + and - to those on the gel. Cover and connect to power source.

4. Perform electrophoresis at 50 volts for 35 minutes.

5. After electrophoresis is complete, remove the gel from the electrophoresis cell and place it in the gel frame. , then put it in the gel hole goo.

6. Place in fixative solution for 5 minutes.

7. Remove from the fixative and dry at 70°C for about 20 minutes.

8. Soak the dried gel in the dye for about 3 minutes.

Note: For gels with very high viscosity (i.e. the gels used in Examples 4 and 6), A sensitive dye is required. Violet and blue dyes are comparable.

9. Decolorize for 3 minutes.

10. Thoroughly absorb the gel with gel absorbent paper.

11. Dry for about 5 minutes.

12. Visually observe the pattern.

When comparing each gel of Examples 1-6 with the naked eye, when alginic acid was used, Hb A, HbS, HbC bands are Hb in the corresponding gel without alginate. Compared to A, HbB, and HbC, - showed significantly less exit shape formation.

Naturally, many variations and variants will be suggested to those skilled in the art based on this disclosure. Ru. These are to be understood as falling within the scope of this invention.

1. Gratzer et aL + Journal of Ku Chromatography, Vol. 315-329 (1961).

2. Robinson et al. J. Lab , & C11in.

Med, 50:645-752 (1957).

3. Barwick et al. Biochimica, Engineering, Pioff Biochimica et Biophysica Acta ) 668: 485-429 (1981).

4. Determination of hemoglobin variants (citrate agar) on of Hemoglobin Variants (Citrate Ag ar)).

Corning Medical (Corning MedicaIl) + Metofil Do, Massache Sets, 02052゜5, Titanium ■ Citrate Hemoglobin Treatment Law (TitanIV C1trate Hemoglobin Proced ure) + Helena 1 Laboratory Lease (Heflena Laboratory) es”).

Embodiments of the invention in which exclusive property rights or privileges are claimed are set forth below.

Procedural amendment November 26, 1985 Mr. Michibe Uga, Commissioner of the Patent Office 1.Display of the incident International Patent Application No. PCT/US851004072, Title of Invention Electrophoretic techniques and improved electricity for the separation of hemoglobin variants running gel 3. Person who makes corrections Relationship to the case: Applicant 4. Agent Address: Katomo Building, 3-4-17 Toranomon, Minato-ku, Tokyo 105 Phone (434) No. 0887 (Main) Name (7002) Patent Attorney Nobuyuki Matsunaga 5. Date of amendment order (Spontaneous) 6. Subject of correction Translation of i and brackets in specification and claims 7. Contents of correction Attachments include the engravings, the specifications, and the translations of the scope of the request. ( ・However, there is no change in the content) 8. Attachment summary of attached documents One copy of each international search report with a translated version of the written description and claims AN'NEX To' L, E! NTERNATZONAL 5EARC)! aE? ORT vNN JP-A-61500810 (5)

Claims (20)

[Claims] 1. An electrophoresis gel having a pH of about 5.5 to about 6.5, comprising: (a) agar, agar; polysaccharides selected from the group consisting of loin, and mixtures thereof; (b) buffer; (c) a compound selected from the group consisting of alginates and alginate esters; An electrophoresis gel comprising: 2. Is the above salt a group consisting of lithium, sodium, potassium and ammonium? The above alginate ester is selected from polypropylene glycol containing up to 80%. The electrophoretic gel according to claim 1, which is esterified. 3. The electrophoresis gel according to claim 1, wherein the gel is agarose. 4. The above buffers are adipic acid, citric acid, ethylenediaminetetraacetic acid, and glutaric acid. , itaconic acid, maleic acid, malic acid, malonic acid, succinamic acid, succinic acid and tricarballylic acid. The electrophoretic gel according to claim 3. 5. Is the above salt a group consisting of lithium, sodium, potassium and ammonium? The above alginate esters are esterified with up to 80% propylene glycol. 5. The electrophoretic gel according to claim 4, which is gelated. 6. 6. The electrophoretic gel according to claim 5, wherein the salt is sodium. 7. 7. The electrophoretic gel of claim 6 further comprising an effective amount of a preservative. 8. Alkyl polyols having further 2 to 6 carbon atoms and 2 to 4 hydroxyl groups 7. The electrophoretic gel of claim 6, comprising a stabilizing amount of. 9. (a) low electroosmotic agarose of about 0.5 to about 1.2%; (b) about 0.1 to about 1% sodium alginate; (c) an effective amount of a preservative; (d) alkyl polyols having 2 to 6 carbon atoms and 2 to 4 hydroxyl groups; Stabilization amount 7. The electrophoretic gel according to claim 6, comprising: 10. (a) low electroosmotic agarose of about 0.7 to about 1%; and (b) about 0.5 9. The electricity of claim 9 comprising ~0.7% sodium alginate. Running gel. 11. (a) adding at least one sample to the gel; (b) the gel of step (a) above; (c) fixing the electrophoresed gel of step (b) above; ;and (d) a type of electrophoresis comprising drying the fixed gel of step (c); electrophoresis method, characterized in that the gel is an electrophoresis gel according to claim 1 above. Electrophoresis method with characteristics. 12. The group in which the above salts consist of lithium, sodium, potassium and ammonium The above alginate esters are etched with up to 80% propylene glycol. 12. The method of claim 11, wherein the method is stealthized. 13. 12. The method of claim 11, wherein said gel is agarose. 14. The above buffer solutions are adipic acid, citric acid, ethylenediaminetetraacetic acid, and glutaric acid. acid, itaconic acid, maleic acid, malic acid, malonic acid, succinamic acid, succinic acid The person according to claim 13 selected from the group consisting of acid and tricarballylic acid. Law. 15. The salt consists of lithium, sodium, potassium, and ammonium selected from the group, wherein the above alginate esters contain up to 80% propylene glycol. 15. The method according to claim 14, wherein the ester is esterified. 16. 16. The method of claim 15, wherein said salt is sodium. 17. Claim 16: The electrophoretic gel further contains an effective amount of a preservative. How to put it on. 18. The above electrophoretic gel also contains 2 to 6 carbon atoms and 2 to 4 water atoms. The method according to claim 16, comprising a stabilizing amount of an alkyl polyol having acid groups. . 19. The above electrophoresis gel is (a) low electroosmotic agarose from about 0.5 to about 1.2%; and (b) from about 0.1 to about 1.2%; 17. The method of claim 16, comprising about 1% sodium alginate. 20. The above electrophoresis gel (a) low electroosmotic agarose from about 0.7 to about 1%; and (b) from about 0.5 to about 0 ．． 17. The method of claim 16, comprising 7% sodium alginate.