Agarose is a kind oforganic compound, chemical formula Ctwenty-fourHthirty-eightOnineteen, is a white or yellow bead shaped gel particle or powder, which is a linear polymer,Basic structureIt is 1,3 linked beta-D-galactose3,6 connected with 1,4-Internal ether-L-galactoseAlternately connectedLong chain[1-2]。
Agar pectin is a heterogeneous mixture composed of many smaller molecules.Agarose is generally dissolved when heated to above 90 ℃ in water, and forms a goodSemisolidThis is the main feature and basis of its multiple uses.agarose gelPerformance is usually usedgel strength express.The higher the strength, the better the gel performance.
Chinese name
agarose[3]
Foreign name
Agarose[3]
Alias
Agarose、Agarose
chemical formula
Ctwenty-fourHthirty-eightOnineteen[3]
molecular weight
six hundred and thirty point five four seven one
CAS login number
9012-36-6[3]
EINECS login number
232-731-8[3]
Melting point
260-481.5℃
Boiling point
993.9 ℃（±65.0 °C at 760 mmHg）
Density
1.8 g/cm³（±0.1 g/cm3）
Appearance
White or yellow bead shaped gel particles or powder
Agarose, abbreviated as AG, is a neutral component of agar without charge, also translated asAgarVegetable or agarose.AgaroseChemical structureIt is β connected by 1,3-D-galactose3,6 connected with 1,4-Internal ether-L-galactoseAlternately connectedLong chainconstitute[1-2]。
originateRed algaeThe main component is polygalactose, of which about 70% is agarose and 30% isBranched chainAgarose.Agarose warelinear structure , by D-GalactoseAnd 3,6-dehydrated galactose form repetitions alternately by linking beta-1,4 and α - 1,3disaccharideCompany.Branched agarose separates from the beta-1,3 bond.Use hot water fromseaweedThe extracted material contains about 40% agar.Commercially available agar (commonly known asChinese cabbage）It is usually in the form of sheet or loose rope, and is usually used as edible gum, drug packaging agent or bacteriaculture mediumuse.Pure agarose is often used in biologyChemical LaboratoryAs a part of electrophoresis, chromatography and other technologiesSemisolidSupporters for biomacromolecules orSmall moleculeSeparation and analysis of substances.
agar, its English name is agar.The word comes fromMalay"Agar agar" means jelly.Also known as agarVegetable bird's nest、Frozen powder, is a class ofCauliflowerAnd other redalgae（Rhodophyceae）Plant extractComing outAlginateIt has a history of more than three hundred years (1658 years) in China and Japan.The Chinese are also called foreign vegetables or frozen powder;Taiwan calls it Cui Yan, andBird's nestThe texture is similar.The Japanese call itCold weather(kanten), because it is harvested in winter.
Agar, as a unique food, has a long history in China.It is composed of large marine algae, cauliflowerLaver, Jiangli, etc., are widely used in food, biochemical and other industries.We often eatjelly, ice creamCakes and Pastries, fudge, canmixed congeeBoth contain agar.Shandong, Fujian, GuangdongHainanEtc. have superiorocean resourcesandclimatic conditions , suitable for largeMarine algaeIt is the main area for agar production, and its products are sold all over the world.
Agar is composed of agarose andagarIt is composed of pectin.Agarose is a linear polymer, and agar pectin is a heterogeneous mixture composed of many smaller molecules.TheirStructural similarity, but with sulfate andcarboxylComponents and gel ability are poor.
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1. Calculation of drainage parametersreference value(XlogP): None
Agarose is generally dissolved in water when heated to above 90 ℃, and forms a good semi-solid gel when the temperature drops to 35-40 ℃, which is the main feature and basis of its multiple uses.agarose gelPerformance is usually usedgel strength express.The higher the strength, the better the gel performance.The strength of high-quality agarose is usually more than 1200 g/cm2 (1% gel concentration).The gelativity of agarose is caused by the existence of hydrogen bond. Any factor that can destroy the hydrogen bond can lead to the destruction of gelativity.Agarose hasHydrophilicity, and there is almost no charged groupBiomacromoleculeIt rarely causes denaturation and adsorption and is an ideal inert carrier.Agar pectin should be removed as much as possible during the preparation of agarose, otherwise there may be very little agaroseSulfate radicalandpyruvic acidSubstitution of ionized groups will cause electricityIntrogression(EEO), electroosmosis has an effect on the movement of particles.The agarose sulfate content of good quality is relatively low, usually below 0.2%, and the electroosmosis is relatively small, usually below 0.13.This is why agarose is so much more expensive than agar.
In 1937, agarose was separated from agar for the first time from wild wood, but it was not until 1961 that Hjertin first discovered the excellent agarosePerformanceLater, it attracted more and more attention and began toindustrial production 。Agarose is widely used in clinical assay, biochemical analysis and biologyMacromolecular substancesAnd other fields.
purpose
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Agar and agarose have specialGellingProperties, especially remarkable stability, hysteresis andHysteresisIt is easy to absorb water and has special stabilizing effect;It has been widely used in food, medicine, chemical industry, textile, national defense and other fields. According to incomplete statistics, agar and agarose have more than 1000 uses, which is internationally known as "novel East Asian products".stayfood industryCan be used to produce: crystalFudge, shaped soft candy, aquatic productsmeat can、Fruit juice beverage、Fruit pulp beverage、Rice WineBeverages, dairy drinks, boutiques, dairy cakes.
In short, remove the agar pectin from the agar, and the remaining part is agarose.
Electrophoretic technique
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characteristic
Agar is a kind ofPolysaccharide, mainly composed of agarose (about 80%) andagarThe composition of adhesive.Agarose is composed ofGalactoseAnd its derivativesNeutral substance, not charged, andAgar gelIt is a kind ofSulfate radicalAnd carboxyl groups. Because these groups are charged, they can produce strong electroosmosis under the action of electric field. In addition, sulfate can interact with someproteinAnd affect the electrophoresis speed and separation effect.Therefore, agarose is often used as electrophoresis supportPlate electrophoresisIts advantages are as follows.
（1）agarose gel electrophoresisThe operation is simple and the electrophoresis speed is fast. The sample can be electrophoresis without prior treatment.
(2) Agarose gel has uniform structure,water contentLarge (about 98%~99%), approximateFree electrophoresisThe sample diffuses more freely than the current, and has very little adsorption on the sample, soElectrophoretic mapClear, high resolution,RepeatabilityOK.
(3) Agarose transparentUltraviolet absorption, electrophoresis process and results can be directly usedultravioletLamp detection and quantitative measurement.
(4) After electrophoresis, the zone is easy to stain, and the sample is easy to elute, which is convenient for quantitative determination.makeDry filmIt can be stored for a long time.
Agarose is often used as electrophoresis support to separate protein andisozyme。Agarose electrophoresis andImmunochemistryCombine and develop intoImmunoelectrophoresisTechnology can identify complex systems that cannot be identified by other methods. Due to the establishment of ultramicro technology, 0.1 ug protein can be detected.
Agarose gel electrophoresis is also commonly used to isolate and identify nucleic acids, such asDNAIdentification, production of DNA restriction endonuclease map, etc.Because this method is easy to operate and the equipment is simpleSample sizeLess,ResolutionHigh, has becomegenetic engineeringOne of the commonly used experimental methods in research.
DNA electrophoresis
The separation of nucleic acids by agarose gel electrophoresis is mainly based on their relative molecular weight and molecular configuration, and is also closely related to the gel concentration.
1. Nucleic acidMolecular sizeRelationship with agarose concentration
（1）DNA moleculeThe size of DNA fragments in the gel, the migration distance of DNA fragments（mobility）AndBase pairOflogarithmIt is inversely proportional, so the size of the unknown segment can be measured by comparing the distance of the standard with known size and the distance of the unknown segment.But when the size of DNA molecule exceeds 20kb, it is difficult to separate them by ordinary agarose gel.At this time, the mobility of electrophoresis is no longer dependent on the molecular size. Therefore, when using agarose gel electrophoresis to separate DNA, the molecular size should not exceed this value.
(2) The concentration of agarose is shown in the following table. DNA of different sizes needs to be processed with agarose gel of different concentrationsElectrophoretic separation。
Table Agarose concentration and DNA separation range
Linear DNA size/kb60-520-110-0.87-0.56-0.44-0.23-0.1
2. Relationship between nucleic acid configuration and agarose gel electrophoresis
DNA with different configurationsMovement speedThe order is: covalently closed circular,cccDNA）Linear DNA Open loopDouble chainCircular DNA。When the concentration of agarose is too high, circular DNA (generally spherical) cannot enter the gel,Relative mobilityIs 0 (Rm=0), while straight lines of the same sizeDouble stranded DNA(rigid rod) can be longAxis directionForward (Rm>0), it can be seen that the relative mobility of the three configurations mainly depends on the gel concentration, but at the same time, it is also affected byelectric currentStrengthBufferionic strengthAnd so on.
For separationnucleic acidAgarose gel electrophoresis can be divided into vertical type and horizontal type (plate type).In horizontal electrophoresis, the gel plate is completely immersed in 1-2mm of electrode buffer solution, so it is also called submersible type.The latter is more used because it is convenient to make glue and add samples,electrophoresis tankIt is simple, easy to make, and can prepare gel plates of different specifications according to needs, saving gel, so it is more popular.
(2) Buffer system
In the absence of ions, the current is too small and DNA migration is slow;On the contrary, the buffer solution with high ionic strength has a large amount of currentThermogenesis, in serious cases, it will cause glue melting and DNAdenaturation。
frequently-usedElectrophoresis bufferyesEDTA(pH8.0) andTris-acetic acid（TAE）, Tris boric acid (TBE) or Tris-phosphoric acid（TPE）The concentration is about 50mmol/L (pH 7.5~7.8).Electrophoresis buffer is generally prepared into a thick stock solution, which is diluted to the required multiple when used temporarily.
TAEBuffer capacityLow, the latter two have high enough buffer capacity, so they are more commonly used.TBEConcentrated solutionLong term storage will lead to precipitation. To avoid this disadvantage, store 5 × solution at room temperature, and dilute 10 times of 0.5 × working solution to provide sufficient buffer capacity.
(3) Preparation of gel
Diluted electrode buffer solution is used as solvent, and boiling water bath orMicrowave OvenPrepare a certain concentration ofSol, fill the horizontal plastic frame or vertical plastic film, insert the comb,Natural cooling。
(4) Sample preparation and sampling
The DNA sample was dissolved in an appropriate amount of Tris EDTA buffer, containing 0.25%Bromophenol blueOr other instructionsdyestuff, containing 10% - 15%sucroseOr 5%~10%glycerolTo increase its specific gravity and concentrate the sample.2.5% Ficoll can be used instead to avoid that sucrose or glycerin may cause U-shaped bands in electrophoresis results（Polysaccharide）Replace sucrose or glycerin.
(5) Electrophoresis
Agarose gel separationmacromoleculeThe results of DNA experimental conditions showed that the separation effect was better at low concentration and low voltage.Under low voltageElectrophoretic mobilityIs the same as the applied voltageProportional。But, inelectric field intensityWhen increasing, the increase of mobility of larger DNA fragments is relatively small.Therefore, with the increase of voltage, the resolution of electrophoresis decreasesMaximum resolutionThe electric field strength should not be higher than 5V/cm.
Electrophoretic systemThe temperature has no significant effect on the electrophoretic behavior of DNA in agarose gel.Generally, electrophoresis is carried out at room temperature. Only when the gel concentration is lower than 0.5%, electrophoresis can be carried out at 4 ℃ to increase the gel hardness.
Biochemistry and Molecular BiologyThe research work ofMolecular hybridizationHowever, agarose is not suitable for hybridization. In 1975, Southern created a method to transfer DNA bands in situ to nitrocellulose membrane (NC membrane) for hybridization, which is called Southern blotting.Later, Alwine et al. used similar methods toRNA imprinting, nicknamedNorthern imprintIn 1979, Towbin et al designed to transfer protein from gel toNitrocellulose membraneDevice, which willProtein transferTo the membrane, and then to the corresponding antibodyligandReaction, nicknamed Western imprinting, is a device that makes membrane, gel, filter paper, etcsandwich biscuitsLow voltage and high current electrophoresis were used to complete the transfer.1982 Reinhart et alElectricity consumptionThe transfer method willIsoelectric focusingThe post protein zone is transferred from the gel to a specific membrane, called Eastern blotting.
There are many kinds of nucleic acids and proteins at home and abroadImprint transferOfElectrophoresis deviceFor sale, the imprint transfer is fast, efficient, repeatable and widely used.polyacrylamide gelIt can also be used for imprint transfer electrophoresis, but when transferring proteins, the gel must not containSDS、ureaetc.Denaturant。For transfer electrophoresisSupporting membraneThere are also many options, which have been used in recent yearsNylon membraneMore because of nylon filmMechanical propertiesGood, baking is not brittle, and it is more convenient to use than nitrocellulose film.
When imprinting transfer electrophoresis is carried out, attention should be paid to the low ionic strength of the buffer solution, the pH should be far away from pI, so that the protein has more charges. Generally, Tris buffer system with good stability is used.Also note that there can be bubbles between the gel and the support membrane.Properly increasing the voltage or current can improve the transfer speed, but it will also increase theheat effect, so the voltage or current must not be too high.
gel electrophoresis
Generally, agarose gel electrophoresis can only separate DNA smaller than 20kb.This is because in agarose gelEffective diameterWhen the gel pore size is exceeded, the DNA is forced to deform and squeeze through under the action of electric fieldSieve apertureAnd straighten along the swimming direction, so the molecular size has little effect on mobility.Change when soElectric field direction, the DNA molecule must change itsconformationStraighten along the new swimming direction, and the turning time is closely related to the size of DNA molecules.In 1983, Schwartz et alRelaxation time(extrapolated to 0Detention time）The characteristics related to the size of DNA molecule, designedPulsed electric fieldGradient gel, alternating two vertical directionsNon-uniform electric fieldThe direction of DNA molecules in the gel is constantly changed, so that DNA is separated according to the molecular size.Later, Carle and others improvedElectrophoretic techniqueIt was also found that the periodic inversion of the electric field can also make macromolecular DNA separate by electrophoresis.The electrophoresis system is composed of a horizontal electrophoresis tank and two groups of independent and mutually perpendicular electrodes. The negative electrode of one group is N, and the positive electrode is S;The negative pole of the other group is W, and the positive pole is E.a blocksquareAgarose gel plate (10cm * 10cm or 20cm * 20cm) is placed in the center at 45 degrees.Electric field atN-SAnd W-E alternately.The time of electric field alternation is related to the size of DNA molecule to be separated.
During electrophoresis, DNA molecules are in an alternating electric field with continuous intervals.First move to the S pole, then change to the E pole.At every timeElectric field directionWhen changing, the DNA molecule will have a certain time to relax, change the shape and migration direction.Only when the DNA molecule reaches a certain configuration can it continue to move forward.The net movement direction of DNA molecule is perpendicular to the sampling line, so that each component in the sample is along the sameLaneForming their own zones.Alternating pulse electrophoresis can effectively separate millions of base pairs of macromolecular DNA.The angle and pulse time between the electrodes of the newer instrument can be adjusted, which is more convenient to use.
