KR100663888B1 - The solution for separation of mitochondria and separation method of mitochondria using the solution - Google Patents

Abstract

The present invention provides a mitochondria from animal tissues such as hepes (HEPES), sucrose, D- mannitol, BSA (eda) (EDTA), leupeptin, pepstein-A, PMSF (PMSF) and In the mitochondrial separation solution and mitochondria separation, characterized in that it comprises a Diti (DTT), it provides a separation method of the mitochondria, characterized in that using the solution.

When the solution and method of the present invention are used, not only the target mitochondria can be separated and extracted by separating and removing other substances in the cell, but also the production efficiency is much better than that of the known art.

Mitochondria, centrifugation

Description

The solution for separation of mitochondria and separation method of mitochondria using the solution

1 is a flow chart showing the mitochondrial pure separation process of the present invention.

2 and 3 are photographs showing the pure separation state of mitochondria separated by the method of the present invention.

The present invention relates to a solution used for separating mitochondria from animal tissues and to a method for separating mitochondria using the same.

Currently mitochondria separation from animal tissue,

"(1) Tissues are removed and the remaining blood is removed cleanly.

(2) Finely slice the tissue with scissors and grind it by adding sucrose solution. At this time, while reciprocating carefully at a speed of 400rpm while maintaining 2 ~ 4 ℃, to prepare a tissue homogenate.

(3) Take out all the homogenate and centrifuge for 10 minutes at 750 × g, then collect the supernatant and centrifuge again at 15,000 × g.

(4) The sucrose solution is added to the precipitate to dissolve it, and then the mitochondrial fraction is separated by sucrose concentration gradient centrifugation.

(5) The obtained mitochondrial fraction was diluted in sucrose solution and centrifuged at 15,000 × g '.

However, the typical mitochondrial separation method has a serious problem of being easily contaminated by little plasma membrane or microsomal.

The ease of mitochondrial pure separation is critical for mitochondrial-related disease research and therapeutic target discovery. However, the mitochondrial pure separation technique, which has been performed in the past, has been difficult to distinguish between mitochondria itself and its protein material due to contamination of mitochondria and other substances in the cell, especially proteins and genes.

Accordingly, an object of the present invention is to provide a novel separation method capable of preventing the contamination of other substances in the cell, that is, known to the known technology, and separating the maximum amount of mitochondria.

In order to achieve the above object, the present invention is Hepes (HEPES), sucrose, D- mannitol, BSA (BSA), EDTA (EDTA), leupetin, pepstein A, PMSF and Diti ( In the separation of mitochondria solution and mitochondria characterized in that it comprises a DTT), it provides a method for separating mitochondria, characterized in that using the solution.

Hereinafter, the present invention will be described in more detail.

In the present invention, hepes (HEPES = N-2- H ydroxy E thyl P iperazine-N'-2- E thane S ulfonic acid), sucrose, D- mannitol, BSA (BSA = B ovine S erum A lbumin), tieyi (EDTA = E thylene D iamine T etra a ceticacid), leupeptin (leupeptin), Pep stein a (pepstain a), piem eseuepeu (PMSF = P henyl M ethyl S ulfonyl F luoride) and di entity (DTT = D i T hio T hreitol ) provides a solution for the separation of mitochondria characterized in that it comprises.

The composition ratio of the composition solution is preferably 2 mM Hepes, 0.7 M sucrose, 0.21 M D-mannitol, 73 μM BC, 0.1 mM IDT, 1 μg / mL leupetin, 1 μg / mL pepstein A, 0.1 mM PMS And 0.1 mM Diti, and the pH is preferably 7.4 (hereinafter, the solution thus prepared is referred to as "H-solution" for convenience).

The mitochondrial separation solution is used as a buffer in the separation of the mitochondria, but serves to effectively discharge the contaminants into the solution, but does not affect the mitochondria membrane can effectively separate the mitochondria.

In addition, the present invention provides a method for separating mitochondria, using the above-described solution as a buffer in the separation of mitochondria. Separation of the mitochondria may be carried out using a known method, but preferably, the tissue is weighed and recorded, and the blood is immediately removed by adding H-solution; Transferring the blood-depleted tissue to a blender, adding H-solution and then grinding to obtain a suspension (2); Suspension Centrifuging (3); Separating the supernatant including the mitochondria after centrifugation (4); Centrifuging the collected supernatant and removing the supernatant (5); After the supernatant was removed, the pellet was resuspended in H-solution. Centrifuging (6); After centrifugation, the upper pellets are removed by dissolving with H-solution, and separating the mitochondrial-containing pellets strongly condensed in the lower layer (7). Mitochondria-containing pellets separated as described above can be stored for one year at -20 ° C for preservation.

At this time, to increase the yield of recovered mitochondria, more preferably the H-solution added in step (2) is twice the volume of the weight of the tissue measured in step (1) (e.g., 1 kg weight of tissue In the case of H-solution 2 Liter), it is preferable that the grinding process to obtain the suspension of step (2) is performed five times at intervals of 4 minutes.

Further, more preferably, the centrifugation of step (3) is preferably performed for 3 minutes at a speed of 1100 × g, and the centrifugation of step (5) is preferably performed for 20 minutes × 20 minutes.

More preferably, the centrifugation of step (6) is preferably performed at a rate of 20000 × g.

On the other hand, it is preferable to further include the step (8) of using a digitonin-type surfactant to remove the mitoplast (mitoplast) to obtain a mitochondride in a more pure form.

At this time, for even more pure separation, preferably the step (8) of using the surfactant of the digitonine, resuspending the pellet obtained in step (7) in H-solution to prepare a mitochondrial solution (a ); Dissolving digitonin in a solution excluding BSA in H-solution and cooling to prepare a digitonin solution (b); Adding the cooled digitonin solution to the mitochondrial solution prepared in step (a) and then stirring (c); After stirring, removing the supernatant by adding H-solution and centrifugation (D); And resuspending the pellet from which the supernatant has been removed and centrifuging again to remove the pellet containing the mitoplasm, and recovering the supernatant containing the mitochondria (e).

At this time, more preferably, the amount of digitotonin used in the step (b) is preferably 0.11 g per 1 g of mitochondrial protein, and the cooling in the step (b) is preferably performed at 4 ° C.

More preferably, the stirring in the step (c) is preferably performed for 15 minutes, and the amount of H-solution added in the step (d) corresponds to three times the weight of the pellet obtained from the step (7). A volume (eg 3 ml H-solution if weighing 1 g of pellet) is recommended.

In addition, more preferably, the centrifugation in step (d) may be performed for 10 minutes at a rate of 10000 × g.

On the other hand, the present invention in order to separate the trapped mitochondria (trapped mitochondria) that is not separated into the supernatant in the step (4) above, the supernatant obtained in step (4) is separated and the remaining pellet is resuspended with H-solution. (A) separating the supernatant containing mitochondria by re-homogenization and centrifugation; The supernatant separated from above Centrifuging (b); (C) resuspending the pellet recovered after centrifugation in H-solution and centrifuging; Resuspending the pellet recovered after centrifugation in H-solution, then centrifuging and discarding the supernatant (d); And discarding the supernatant and resuspending the remaining pellets in H-solution, and then adding the supernatant recovered from centrifugation (e) to the " gathered supernatant " Good to do.

By adding the above method, it is possible to recover trapped mitochondria, which is advantageous in that a larger amount of mitochondria can be recovered from the sample.

On the other hand, in order to increase the yield of the recovered mitochondria, more preferably, the centrifugation of step (b) is preferably carried out for 15 minutes at a rate of 6780 × g, the centrifugation of the step (c), 20000 × 10 minutes at a speed of g is recommended.

Further, more preferably, the centrifugation of step (d) may be performed at a speed of 20000 × g, and the centrifugation of step (e) may be performed at a speed of 3000 × g for 3 minutes.

Further, more preferably, after centrifugation of step (e), the supernatant is separated and the remaining pellets are resuspended in H-solution, centrifuged to recover the supernatant, and recovered by centrifugation in step (e). The method may further include mixing with the prepared supernatant.

By resuspending and centrifuging the pellet repeatedly, more trapped mitochondria can be recovered. In the case of many trapped mitochondria, resuspension and centrifugation can be carried out one or more times as necessary.

As described above, the present invention is a method for separating a large amount of pure mitochondria from the tissue, the method for separating mitochondria of the present invention minimizes contamination by a little plasma membrane or microsomal, in particular And optionally separating the mitoplasts to minimize contamination of organelles such as ribosomes.

Therefore, compared with the conventional method for separating mitochondria, it is possible to effectively separate a large amount of pure mitochondria. In addition, the present invention allows for the pure separation of large amounts of mitochondria from tissue within 30 minutes.

Meanwhile, the mitochondrial pure separation technology of the present invention is applicable to cell samples of various species having mitochondria in cells such as humans, guinea pigs, rabbits, mice, mice, fishes, crustaceans, amphibians, and microbial species.

The mitochondrial pure separation technology of the present invention provides an advantage that can completely perform its own function without damaging the mitochondrial bilayer and surface proteins.

 Figure 1 shows a flow chart showing the mitochondrial pure separation process of the present invention.

Or more, the configuration of the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited only to the following examples.

Example 1 Isolation of Mitochondria According to the Present Invention

1. Weigh and record the liver tissues of rats, immediately below the solution prepared in the present invention, referred to as 'H-solution'; 2 mM Hepes, pH 7.4, 0.7 M sucrose, 0.21 M D-mannitol, 73 μM BSA, 0.1 mM IDTA, 1 μg / mL leupeptin, 1 μg / mL pepsin A (pepstain A), 0.1 mM PMSF, 0.1 mM Diti (DTT) was used to remove blood.

2. The suspension was prepared by transferring the tissue to a blender, adding a volume of H-solution corresponding to twice the weight and then grinding five times at four minute intervals.

3. The suspension was centrifuged for 1100 x g for 3 minutes.

4. Thereafter, the supernatant containing the mitochondria was carefully transferred. The remaining pellets contain intact liver, nucleus, and trapped mitochondria.

5. The collected supernatant was centrifuged for 20 minutes at 20000 g, and the supernatant was removed. The light layer, i.e. the material at the top of the pellet, is easily broken down by H-solution into the broken mitochondria.

6. The bottom heavy heave was resuspended in H-solution and centrifuged at 20000 × g. As before, light upper pellets were melted and removed using H-solution, and tightly packed pellets containing mitochondria were stored at -20 ° C, which can be stored for one year.

Example 2 Separation of Mitochondria According to the Present Invention

1. Weigh and record the liver tissues of rats, immediately below the solution prepared in the present invention, referred to as 'H-solution'; 2 mM Hepes, pH 7.4, 0.7 M sucrose, 0.21 M D-mannitol, 73 μM BSA, 0.1 mM IDTA, 1 μg / mL leupeptin, 1 μg / mL pepsin A (pepstain A), 0.1 mM PMSF, 0.1 mM Diti (DTT)) to remove blood.

2. The suspension was prepared by transferring the tissue to a blender, adding a volume of H-solution corresponding to twice the weight and then grinding five times at four minute intervals.

3. The suspension was centrifuged for 1100 x g for 3 minutes.

4. Thereafter, the supernatant containing the mitochondria was carefully transferred. The remaining pellets contain intact liver, nucleus, and trapped mitochondria.

5. To extract the trapped mitochondria, the pellet was resuspended in H-solution, re-homogenized and centrifuged. This supernatant containing mitochondria is called post-nuclear supernant (PNS).

6. The pellet was recovered by centrifuging the PNS for 6780 × g for 15 minutes.

7. The recovered pellet was resuspended in H-solution and centrifuged at 20000xg for 10 minutes. Supernatants were discarded because they contained cellular microsomes and the like. The pellet was resuspended in H-solution to remove the trapped microsomes, centrifuged at 20000 x g and the supernatant was discarded.

8. The large-microsome free mitochondrial pellet was resuspended in H-solution, and then the supernatant was separated by centrifugation at 3000 x g for 3 minutes. At this time, the pellet is made of residual nuclei, plasma membrane, and lysosome. And so on.

9. The supernatant was transferred to a new tube, and the pellet was resuspended in H-solution, centrifuged and added to an electric tube to collect the supernatant.

10. The supernatant collected from the above "9" and "4" was centrifuged for 20 minutes at 20 000 g, and the supernatant was removed. The light layer, i.e. the material at the top of the pellet, is easily broken down by H-solution into the broken mitochondria.

11. The lower heavy pellets were resuspended in H-solution and then centrifuged at 20000 × g. The light upper pellets, as before, were removed by melting with H-solution and the tightly packed pellets containing mitochondria were stored at -20 ° C.

Example 3 Isolation of Mitochondria According to the Present Invention

1. Weigh and record the liver tissues of rats, immediately below the solution prepared in the present invention, referred to as 'H-solution'; 2 mM Hepes, pH 7.4, 0.7 M sucrose, 0.21 M D-mannitol, 73 μM BSA, 0.1 mM IDTA, 1 μg / mL leupeptin, 1 μg / mL pepsin A (pepstain A), 0.1 mM PMSF, 0.1 mM Diti (DTT)) to remove blood.

2. The suspension was prepared by transferring the tissue to a blender, adding a volume of H-solution corresponding to twice the weight and then grinding five times at four minute intervals.

3. The suspension was centrifuged for 1100 x g for 3 minutes.

4. Thereafter, the supernatant containing the mitochondria was carefully transferred. The remaining pellets contain intact liver, nucleus, and trapped mitochondria.

5. To extract the trapped mitochondria, the pellet was resuspended in H-solution, re-homogenized and centrifuged. This supernatant containing mitochondria is called post-nuclear supernant (PNS).

6. The pellet was recovered by centrifuging the PNS for 6780 × g for 15 minutes.

7. The recovered pellet was resuspended in H-solution and centrifuged for 10 minutes at 20000 g. Supernatants were discarded because they contained cellular microsomes and the like. The pellet was resuspended in H-solution to remove the trapped microsomes, centrifuged at 20000 x g and the supernatant was discarded.

8. The large-microsome free mitochondrial pellet was resuspended in H-solution, and then the supernatant was separated by centrifugation at 3000 x g for 3 minutes. At this time, the pellet is made of residual nuclei, plasma membrane, and lysosome. And so on.

9. The supernatant was transferred to a new tube, and the pellet was resuspended in H-solution, centrifuged, and added to an electric tube to collect.

10. The supernatant collected from the above "9" and "4" was centrifuged for 20 minutes at 20 000 g, and the supernatant was removed. The light layer, i.e. the material at the top of the pellet, is easily broken down by H-solution into the broken mitochondria.

11. The lower heavy pellets were resuspended in H-solution and then centrifuged at 20000 × g. Light upper pellets, as before, were dissolved by H-solution to remove and recovered the tightly packed pellets containing mitochondria.

On the other hand, by using the separation method of the various steps, it was possible to first reduce the contamination by the little plasma membrane (microplasmal) or microsomal (microsomal). However, in order to minimize contamination, a process of separating mitoplast as a contaminant as incomplete mitochondria was further performed as follows.

12. The mitochondrial pellets obtained from step 11 above were resuspended in H-solution. At this time, digittonin (about 0.11 g of digittonin relative to 1 g of mitochondrial pellet weight) was dissolved in about 10 mL of H-solution (H-solution without BSA). The solution to which the digitonin was added was slightly boiled with stirring until it melted, and when it was dissolved, it was left to cool at 4 ° C for 1 hour.

13. The coldly prepared Digitonin solution was added to the mitochondrial solution suspended in "Step 12" and stirred for 15 minutes.

14. Subsequently, centrifuge 10000 × g for 10 minutes by adding H-solution as much as three times the volume of the measured “12” mitochondrial pellets (eg, 3 ml of H-solution for 1 g of pellet). It was. The supernatant is the fraction containing the outer mitochondrial membrane and can be removed or stored if necessary for other experiments.

15. The pellet recovered in step 14 is resuspended in H-solution and centrifuged again. The pellet thus obtained is a fraction containing mitoplasm as a contaminant and the supernatant is a fraction containing pure mitochondria.

Experimental Example 1: Investigation of Purity of Isolated Mitochondria

2 shows an experimental result of observing the presence or absence of a protein using an antibody of the mitochondrial indicator protein (ATPase) and mitochondria, but not in the mitochondria, but in other organs through protein electrophoresis. .

First, the supernatant excluding the mitochondria in lane 1 and the second, electrophoresis of the purified mitochondrial protein.

Cav was not found in the mitochondria but was present in the cell membrane and was not observed in the mitochondrial protein (lane 2). The mitochondrial indicator protein "ATPase" was only observed in the mitochondrial protein.

In the experiment of FIG. 3, the mitochondria were colored using a fluorescent marker (MitoTracker) that selectively reacts with the mitochondria and observed using confocal microscopy. As a result, purely separated mitochondria were observed to be dyed red.

Therefore, from the above results, we found that the mitochondria that we separated were well separated without contamination of other organelles.

As described above, the present invention provides a solution for obtaining mitochondria from animal tissues and a method for purely separating the mitochondria through centrifugation, thereby separating other mitochondria from the cells by separating other mitochondria. The amount also has many superior effects over the methods of the prior art.

Claims (17)

Hepes (HEPES), sucrose, D- mannitol, BSA (EDA), leupetin, pepstein A, PMSF and DTT (DTT) characterized by Solution for the separation of mitochondria. The composition of claim 1, wherein the composition of the solution is Composed of 2 mM Hepes, 0.7 M sucrose, 0.21 M D-mannitol, 73 μM BD, 0.1 mM IDT, 1 μg / mL Lupeptin, 1 μg / mL Pepstein A, 0.1 mM PMS and 0.1 mM Diti pH is a mitochondrial solution, characterized in that 7.4. The separation method of mitochondria, wherein the solution according to claim 1 is used as a buffer. The method of claim 3, wherein the separation, Measuring and recording the tissue weight and immediately removing blood by adding the mitochondrial separation solution of claim 1; Transferring the blood-depleted tissue to the blender, adding the mitochondrial separation solution of claim 1 and then grinding to obtain a suspension (2); Suspension Centrifuging (3); Separating the supernatant including the mitochondria after centrifugation (4); Centrifuging the collected supernatant and removing the supernatant (5); After resuspending the pellet from which the supernatant was removed, Centrifuging (6); And After centrifugation, the upper pellet With the solution of claim 1 And dissolving and removing the mitochondrial-containing pellet (7) strongly condensed in the lower layer. 5. The method of claim 4, wherein the amount of the solution of claim 1 added in step (2) is twice the volume ratio of the tissue weight measured in step (1); 5. The method of claim 4, wherein the grinding process for performing the suspension of step (2) is performed five times at intervals of four minutes. The method of claim 4, wherein the centrifugation of step (3) is performed for 3 minutes at a speed of 1100 x g, and the centrifugation of step (5) is performed for 20 minutes at a speed of 20000 x g. Centrifugation of (6) is the separation method of mitochondria, characterized in that carried out at a rate of 20000 × g. 5. The method of claim 4, further comprising the step (8) of using a digtonin-like surfactant to remove mitoplasm. The method of claim 8, wherein the step (8) of using a digtonin family of surfactants, Resuspending the pellet obtained in step (7) in the solution of claim 1 to prepare a mitochondrial solution (A); Dissolving digitonin in a solution excluding BSA in the solution of claim 1 and cooling to prepare a digitonin solution (b); Adding the cooled digitonin solution to the mitochondrial solution prepared in step (a) and then stirring (c); After stirring, adding the solution of claim 1 to centrifugation to remove the supernatant (d); And Resuspending the pellet from which the supernatant was removed and centrifuging again to remove the pellet containing the mitoplasm, and recovering the supernatant containing the mitochondria (e). Separation method of mitochondria. 10. The method of claim 9, wherein the amount of digitonin used in step (b) is 0.11 g per 1 g of mitochondrial protein. 10. The method for separating mitochondria according to claim 9, wherein the cooling in the step (b) is performed at 4 ° C. 10. The method for separating mitochondria according to claim 9, wherein the amount of the solution of claim 1 added in step (d) is 3% by volume of the pellet weight obtained from step (7). The method for separating mitochondria according to claim 9, wherein the centrifugation in step (d) is performed for 10 minutes at a rate of 10000 × g. The method of claim 4, wherein (A) separating the supernatant containing the mitochondria by separating the supernatant from step (4) and resuspending the remaining pellets with the solution of claim 1, followed by re-homogenization and centrifugation; The supernatant separated above Centrifuging (b); (C) resuspending the pellet recovered after centrifugation with the solution of claim 1 and centrifuging; (D) resuspending the pellet recovered after centrifugation with the solution of claim 1, then centrifuging and discarding the supernatant; And After discarding the supernatant and resuspending the remaining pellets with the solution of claim 1, the supernatant recovered from centrifugation (e) is added to the collected supernatant described in step (5), characterized in that the subsequent steps are carried out. Separation method of mitochondria. The method of claim 14, wherein the centrifugation of step (b) is performed for 15 minutes at a speed of 6780 × g, the centrifugation of step (c) is performed for 10 minutes at a rate of 20000 × g, and the step (d Centrifugation) is carried out at a rate of 20000 × g, the centrifugation of step (e) is performed for 3 minutes at a rate of 3000 × g. 15. The method according to claim 14, wherein after centrifugation of step (e), the supernatant is separated and the remaining pellet is resuspended with the solution of claim 1, followed by centrifugation to recover the supernatant, followed by centrifugation in step (e). Separation method of the mitochondria, characterized in that it further comprises the step of mixing with the recovered supernatant. Mitochondria separation product separated by the method of claim 4 using the solution of claim 1.

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