Aerogel is also called jelly. Colloidal particles or polymers in sol or solution are interconnected to form a spatial network structure under certain conditions, and the gaps in the structure are filled with liquid (or gas in xerogel) as a dispersion medium. This special dispersion system is called gel. No liquidity. The interior usually contains a lot of liquid. For example, the water content of blood gel and agar can reach above 99%. It can be divided into elastic gel and brittle gel. After losing the dispersion medium, the volume of elastic gel decreases obviously, and when absorbing the dispersion medium again, the volume expands again, such as gelatin. When the brittle gel loses or reabsorbs the dispersion medium, its shape and volume remain unchanged, such as silica gel. The process of forming gel from solution or sol is called gelation.

[Edit this paragraph] Biology and gel

The downstream purification objects of biomolecules generally include protein, enzymes, recombinant proteins, monoclonal antibodies, antibodies and antigens, peptides, viruses, nucleic acids and so on. Before purification, it is necessary to determine the physical and chemical characteristics of biomolecules, and then choose the most effective purification process through experiments.

1. determination-molecular weight, PI

When the molecular weight, PI and other physical properties of the target protein are not clear, it can be determined by PAGE electrophoresis or chromatography. Superose HR packed column with wide separation range is very suitable for determining the molecular weight of unknown protein. In several test tubes containing different PH buffers, PI can be easily measured with a small amount of ion exchange media, and the optimal PH of the purified buffer can be selected.

2. Selective chromatography

If you don't know much about the characteristics or sample composition of the target protein, you can try several different purification methods:

1) Using the most common gel filtration method, select the media with wide separation range, such as Superose and Sephacryl HR, and divide the sample into different components according to the molecular weight.

2) combining the target protein with an affinity chromatography medium containing a specific ligand or antibody. Various activated coupling media can also be used to couple the substrate and receptor of the target protein, and then used to bind the target protein. High purity samples can be obtained in one step.

3] Large samples are usually concentrated and purified by ion exchange chromatography. Samples eluted with high salt can be purified by hydrophobic chromatography. Hydrophobic chromatography uses the principle of high salt adsorption and low salt elution, and the eluted sample can be directly subjected to adsorption chromatography such as ion exchange. These two methods are often used alternately in the purification process.

3. Purification-A large number of crude products

In order to avoid column blockage, large particle ion exchange media such as sepharose big beads, sepharoseXL, sepharose fast flow, etc. are generally used when dealing with a large amount of stock solution. The expanded column bed adsorption technology uses various streamlined media to capture protein directly from the fermentation broth containing broken cells or tissue extracts. Centrifugation, ultrafiltration and preliminary purification are combined into one. Improve the recovery rate and shorten the purification period.

4. Purification-ammonium sulfate precipitation method is often used for preliminary purification of samples, and the treated samples are in high salt state, which is very suitable for direct hydrophobic chromatography. For ion exchange, it is necessary to desalt with Sephadex G-25 first. Hydrophobic chromatography is a relatively new technology, which is gradually integrated into various production processes with the increasing variety of media. Hitrap HIC test kit and RESOURCE HIC test kit can be used to select the most suitable medium and the best purification conditions among eight hydrophobic media. Samples eluted with low salt can be slightly diluted or directly subjected to other adsorption chromatography.

5. Pure water-sugar molecules

Immobilized lectins, such as concanavalin, peanut, barley, etc. Can bind carbohydrate residues, and is very suitable for separating glycated cell membrane components, cells and even subcellular organelles, and purifying glycoproteins. Two Sepharose 6MB affinity chromatography media with lectins are designed to capture whole cells or large complexes, such as membrane capsules.

6. Purification-membrane protein

Detergents are often used to separate membrane proteins to maintain their activity. The ion detergent should be selected with the opposite charge to the target protein, so as to avoid competing with the target protein for exchange medium in the ion exchange process, thus removing the detergent. Nonionic detergents can be removed by hydrophobic chromatography.

7. Purification-Most monoclonal antibodies and antigen * monoclonal antibodies are IgG. The main sources are ascites and supernatant of fusion tumor culture. Ascites contains a lot of albumin, transferrin and host antibody. Mabselect, g protein and a protein have specific affinity to the Fc region of IgG, and can purify IgG from different sources in one step. Serum complement such as calf serum can be pretreated with G protein first, and IgG can be removed before culture. Mabselect and rProtein A Sepharose FF in recombinant protein A medium have higher load and specificity for IgG, and less population shedding. Dropped protein A can be easily removed by ion exchange Q Sepharose HP or gel filtration Superdex 200.

* hydrophobic chromatography phenyl agarose gel HP is also suitable for purifying IgG. Host antibody and contaminated IgG can be removed by gel filtration Superdex 200 in fine purification.

* The most effective way to purify IgG antigen is to couple IgG with activated coupling media such as CNBr and NHs-activated Sepharose FF, and then further obtain IgG antigen.

*HiTrap IgM is a monoclonal antibody used to purify fused tumor cells, and the binding amount is 5mg IgM. HiTrap IgY is specially used to purify IgY, and the binding amount is 100mg of pure IgY.

8. Purification-The concept of purification should have been integrated into the design and construction of recombinant protein. Most samples are mixed with broken cells or dissolved products, so the expanded column bed adsorption technology STREAMLINE is very suitable for coarse separation. Amersham biosciences provides three fusion systems of rapid expression and one-step purification.

1] GST fusion vector makes the protein to be expressed express together with glutathione S transferase, then purified by affinity chromatography with glutathione agarose 4B, and then cleaved by thrombin or factor Xa.

2. Fusion Vector of Protein A The protein to be expressed was fused with IgG binding site of Protein A, and purified by IgG agarose 6 FF.

2] Fusion protein with histidine tag can chelate Ni2+ metal with chelating agarose FF, and the fusion protein can be chelated by histidine under normal or denatured conditions (8M urea). HisTrap kit provides a complete set of purification methods for His-Tag protein.

9. Purification-Inclusion Body Protein

Inclusion body proteins often need to be dissolved in 6M guanidine hydrochloride or 8M urea. Suprose12 and Sepharose 6FF gel filtration media with high chemical stability are suitable for purification under denaturing conditions. Denatured and purified protein needs renaturation to its natural conformation. Superdex 75, Q Sepharose FF and phenyl Sepharose FF are helpful to the renaturation of inclusion body proteins. The higher the purity of inclusion body protein samples, the better the renaturation effect. SOURCE 30 RPC reversed-phase chromatography medium is very suitable for purifying crude products before renaturation, and can be regenerated by 1MnaOH. The renaturation recovery rate of inclusion body protein purified by this method is obviously improved.

10. Solid-phase renaturation of inclusion body proteins * In recent years, many literatures reported that inclusion body proteins were immobilized (adsorbed) on chromatographic media under denaturing conditions, and various Sepharose FF ion exchange chromatographic media were generally used. After the denaturant was removed, protein was successfully renatured on the culture medium, and then the renatured protein was eluted. Solid-phase renaturation avoids the formation of protein polymer in the general renaturation process, so the renaturation yield is higher, and there is no need to dilute a large number of samples. Renaturation and preliminary purification are combined into one, which greatly saves time and improves the recovery rate.

* Solid-phase renaturation method is also used for direct renaturation and purification of histidine fusion protein expressed as inclusion body by HiTrap chelate metal chromatography; The fusion protein containing multiple lysine was directly renatured and purified by HiTrap heparin affinity chromatography. Both affinity chromatography pre-packed columns can be reused, which is more convenient and durable than general kits.

1 1. Purification of effective components of Chinese herbal medicine

The chemical composition of traditional Chinese medicine is extremely complicated. Traditional Chinese medicines are mostly taken after decoction, and the effective components are mostly hydrophilic components, including alkaloids, flavonoids, anthraquinones, saponins, organic acids, polysaccharides, peptides, protein and so on. Flexible and comprehensive use of a variety of chromatographic methods. Such as ion exchange, molecular sieve and reversed-phase chromatography. Sephadex LH-20 dextran gel has the functions of adsorption chromatography and molecular sieve. For example, if methanol is used to separate flavonoid glycosides, trisaccharides are washed off first, followed by disaccharides, monosaccharides and aglycones. Sephadex LH-20 can use various reagents such as water, ethanol, acetone and chloroform, and is widely used for the separation of various natural products, including alkaloids, glycosides, flavonoids, quinones, lipids, terpenoids and steroids.

* Alkaloids are positively charged in acidic buffer and become salts. HiTrap SP cation exchange column can separate many alkaloids with very similar structures. On the contrary, flavonoids, anthraquinones, saponins and organic acids can be dissolved in alkaline buffer, and the separation effect is better on HiTrap Q anion exchange column.

* Generally, molecular sieves such as Sephadex and Sephacryl are used to purify polysaccharides. If the molecular weight is below 600KD and higher resolution is needed, a new generation of Superdex can be selected. Generally speaking, plants may contain water-soluble, acid-soluble and alkali-soluble polysaccharides. The comprehensive utilization of molecular sieve and ion exchange chromatography is helpful to further obtain the pure products of each component. In addition, polysaccharide drugs need to remove protein, which will cause allergies. The traditional Sevag method needs to be repeated dozens of times to deproteinize butanol. The anion exchange method can quickly remove the residual protein in polysaccharide in one or two steps. SOURCE5, 15, 30RPC reversed-phase chromatography is also very suitable for the detection, separation and amplification preparation of effective components of various traditional Chinese medicines. Because the components of traditional Chinese medicine are very complex, the available range of source reversed-phase chromatography is PH 1- 14, and it can be washed and regenerated with 1M NaOH and 1M HCL. Compared with the traditional silica gel reversed-phase chromatography, it is easier to optimize the process and clean in place, and the service life is longer.

12. purification-peptide

The sources of peptides are natural extracted peptides, synthetic peptides and recombinant peptides. Peptides are easily degraded by enzymes, but they can be renaturated from organic solvents or cosolvents, so they are purified by highly selective reversed-phase chromatography, such as SOURCE 30RPC, SOURCE 15RPC, SOURCE 5RPC or ion-exchanged Minibeads and Monobeads. Superdex peptide HR is a gel filtration pre-packed column specially designed for the purification of peptide molecules, which can be combined with reverse phase chromatography to make a more exquisite peptide map. Peptide molecules can be prepared by ion exchange combined with gel filtration. Multifunctional medical city

13. purification-nucleic acids and viruses

Purification of nucleic acid is used to remove pollutants that affect sequencing or PCR. Nucleic acid can be roughly divided into plasmid DNA, phage DNA and PCR products. Viruses can also be regarded as nucleic acid macromolecules. Like plasmid DNA, we can use Sephacry S- 1000 SF, Superose or Sepharoce 4FF gel filtration media to remove impurities, and then cooperate with Mono Q and SOURCE Q plasma exchange to separate nucleic acids.

14. purification-oligonucleotide monophosphate is widely used in antisense DNA, RNA sequencing, PCR and cDNA synthesis. After synthesis, the by-products of triphenylmethyl-containing oligonucleotides can be removed at PH 12 with anion exchange Mono Q or fast low back pressure source q to avoid agglutination and loss of protective groups. The loading capacity is much higher than that of reversed-phase chromatography, and it can be used for mass preparation. The failed sequence without triphenylmethyl group can be removed by reversed-phase column ProRPC.

15. Desalination and small molecule removal

Gel filtration media Sephadex G 10, G 15, G25, G50, etc. Small molecules are removed with high efficiency, and the treatment capacity can reach 30% of the bed volume. Small molecules below the upper limit of the packing separation range can be easily removed by collecting the eluent in the first column volume of 1/3- 1/2 after injection. Because only small molecules were removed, the column height was above 10cm. The whole process can usually be completed in a few minutes to half an hour. Sephadex G25 series media is specially designed for desalination in protein, and the pre-packed column HiTrap desalination (5ml) can be operated by syringe. HiPrep desalination (26ml) can desalt samples up to 10ml in a few minutes.

16. Vaccine purification The vaccine was purified by gel filtration medium Sepharose 4FF, and the impurity protein in the medium was removed. The treatment capacity can be greater than 10% of the bed volume. The column height is generally 40-70cm, and the whole process takes about half an hour. At present, vaccines produced by this method include hepatitis B, rabies, hemorrhagic fever, influenza, tuberculosis and polio vaccines. Sephacryl S-500HR can be used for small molecular weight vaccines, such as hepatitis A vaccine.

17. analysis of antibiotic polymers

Since 2000 edition, China Pharmacopoeia requires to find out the percentage of polymer in ceftriaxone sodium, and determine it by Sephadex G 10 gel filtration method.

18. purified virus vector for gene therapy

SORRCE 15Q

19. purified plasmid for gene therapy

Q Sepharose XL, SOURCE 15Q, STREAMLINE Q, Sephacryl S500, Plasmidselect, in downstream purification, different chromatographic techniques can be applied to purify biomolecules and remove various pollutants at the same time.

1. Remove-endotoxin

Endotoxin is also called pyrogen. It contains fat A, sugar and protein, and is a composite polymer with negative charge.

The fatty part of endotoxin has strong hydrophobicity. But it will agglutinate under high salt, so hydrophobic chromatography cannot be carried out. Hydrophobic chromatography test kit (17-1349-01) can be used to select the medium that binds to the target protein and remove endotoxin.

Endotoxin is closely bound to anion exchange medium q or DEAE agarose gel Fast Flow. After eluting the target protein, it can be removed by high salt buffer or NaOH.

CNBr or NHS Sepharose FF can be used to couple endotoxin substrates, such as LAL and PMB, and automatically form an affinity chromatography medium to bind endotoxin. Endotoxin is usually a polymer, which can be effectively removed by gel filtration chromatography.

2. Removing nucleic acid from protein.

A large number of nucleic acids increase the viscosity of the sample, enlarge the area, increase the back pressure, and reduce the resolution and flow rate. Drug testing and food testing also have strict restrictions on nucleic acid content.

The problem of nucleic acid expressing protein in cells is particularly serious. Nucleic acid is negatively charged. In the initial purification, a large number of nucleic acids can be removed by using cation exchange media such as Streamline, SP Sepharose Big Beads, SP or CM Sepharose FF, SP SepharoseXL, etc.

Nucleic acid will dissociate from protein under high salt, and hydrophobic chromatography medium is very suitable for binding to target protein, and removing nucleic acid while purifying protein.

Nucleic acid is cut into small pieces by nuclease, which can be easily removed by gel filtration for fine purification.

Step 3 Remove-Viruses and Microorganisms

Viruses and microorganisms will become pathogens and should be reduced as much as possible. Combining different chromatographic techniques, using water for injection and regularly disinfecting and cleaning instruments and gels with NaOH can avoid the increase of pollutants.

Most viruses have lipid shells. Viruses can be inactivated by S/D (solvent/detector) with opposite charge to the target protein, such as Triton and Tween. Then, the target protein can be combined with an appropriate ion exchange medium (such as CM agarose FF) to remove S/D. 。

Other pollutants can change the pH value and ionic strength, so that they can be separated from the target molecules or inactivated. Superdex, a gel filtration medium, and SOURCE, a variety of adsorption media, are good fine purification media, which can remove a variety of trace pollutants.

Gel refers to a mixture with a particle size of 1 angstrom to 10 angstrom. Polymer solution and some sols, under appropriate conditions, the whole system will be transformed into an elastic semi-solid viscous substance, losing fluidity. This phenomenon is called gelation, and the product formed is called gel or jelly.

"Aerogel" means that the dispersion system is gaseous, such as clouds and fog, "solid gel" includes smoky crystals, and "liquid gel" is liquid colloid, such as iron hydroxide colloid.

Example:

Food grade glucomannan gum

Glucomannan gum (also known as konjac gum) is a new type of multi-purpose granular edible gum. The glucomannan introduced here is grape mannan extracted from high-quality konjac, which is processed by advanced technology to remove impurities. The added ingredients do not contain any non-edible chemical components and pigments. Compared with traditional food additives such as agar, pectin and seaweed gum, glucomannan gum is obviously superior to the above-mentioned gums in terms of expansion rate, viscosity, thickening, stability and ease of use. In addition, it has special advantages: under acidic, neutral or alkaline conditions, it can be frozen at room temperature without adding any coagulant, and its gel performance is ideal; Maintaining or strengthening the health care functions of grape mannan such as lowering blood sugar, reducing blood fat and losing weight, and greatly broadening the application range of konjac; Low price and convenient use.

Glucomannan gum can be widely used in food, beverage, medicine, daily chemical, scientific research and other fields. As a substitute for agar, pectin, seaweed gel, etc. , the price is low, and the use cost of the additive can be greatly reduced without changing the original equipment and technology. It is an ideal new gel additive. To meet the needs of different product development, 1. Gel (jelly) type: It can be well mixed with various natural juices, materials and pigments. As a broad-spectrum gel excipient, it is an excellent raw material for making jelly, crystal soft candy and so on. And can also be used as a culture medium bracket without adding any other gum or alkaline components; The gel-forming conditions are random, the cup is completely removed, and the gel has high strength and toughness. Dosage: 0.7 ~ 0.9%. Usage: Swell in proper warm water for 3 ~ 5 minutes, add sugar and various ingredients when boiling and cooling to about 70 degrees, and then cool to room temperature.

2. Culture medium type: instead of agar, it is used as a support for tissue culture of flowers and other plants. The roots of tissue culture seedlings are thick and Miao Zhuang, and the use effect is ideal, and the cost is greatly reduced. Dosage: 0.5 ~ 0.6%. Usage: expand in warm water for 3 ~ 5 minutes, boil and cool. 3. Slurry (tea) drinking type: as a stabilizer and suspending agents, replacing agar and carboxymethyl cellulose. Widely used in granular orange, fruit tea, fruit juice, soybean milk, tremella, eight-treasure porridge and other multiphase suspending agents. (or mixing) to prevent precipitation or stratification. Dosage: about 0. 15 ~ 0.25%. Usage: After fully swelling in proper warm water, add it into the material. Type of frozen products: used for frozen products such as popsicles and ice cream, which can increase expansion, reduce ice crystals, improve heat melting resistance and make products more refreshing. Dosage: about 0. 15 ~ 0.25%. Usage: After fully swelling in proper warm water, add it into the material. 5. Thickening type: used in jam, rice and flour products, it can increase expansion rate, improve toughness and improve shape and taste. It is an ideal substitute for importing locust bean gum. Dosage: about 0.2 ~ 0.3%. Usage: After fully swelling in proper warm water, add it into the material.