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FAQ—Solarbio Fillers

FAQ Documentation—Fillers


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Q01:Can you provide me with the specifications for Dextran Gel G-10, G-15, G-25, G-50, G-75, G-100, G-150, and G-200?
A01:The specifications for these gel fillers are generally universal. You can directly search for "Dextran Gel" to access the comprehensive information.

Q02:What is the shelf life of these fillers?
A02:The shelf life is indicated on the labels. You can request the labels for detailed information. Typically, customers should refer to the information provided on the product labels. Pre-swollen fillers have a longer shelf life and are usually stored in 20% ethanol. Dry powder fillers have a shorter shelf life.

Q03:How should the fillers be stored?
A03:Follow the storage instructions on the label until use. After swelling, adhere to the preservation methods outlined in the product manual. Generally, after swelling and rinsing thoroughly, store the fillers in 20% ethanol. Ensure no freezing occurs during storage. Regularly monitor and replace the liquid to prevent bacterial growth. Promptly add 20% ethanol when the liquid level is low. Keep swollen fillers immersed in liquid to prevent dehydration.

Q04:What is the recommended pretreatment method for Dextran Gel?
A04:It is advisable to soak the gel overnight rather than boiling, as prolonged boiling can damage the structure of the filler.

Q05:What are the differences between D8900 DEAE-32 and C8930 DEAE-52?
A05:Both are weak anion exchange resins. DEAE-32 is in dry powder form, while DEAE-52 is pre-swollen and stored in 20% ethanol.

Q06:How do I choose the appropriate chromatography column?
A06:Our chromatography columns include affinity columns, standard columns, and medium-pressure glass columns. Affinity columns are typically smaller in volume with 50um sieve plates at both ends. Standard and medium-pressure glass columns come in various specifications and can withstand pressure. Choose based on your desired column height and diameter.

Q07:Do you offer pre-packed columns?
A07:Yes, we do. Customization is available according to customer requirements.

Q08:What are equilibration buffers and elution buffers?
A08:For ionic fillers, equilibration buffers and protein sample buffers have the same pH and ionic strength to facilitate protein binding. Elution buffers typically have a higher ionic concentration than equilibration buffers or employ gradient elution to effectively elute proteins.

Q09:What should I do if my sample is colored?
A09:Before loading the sample onto the column, it is recommended to filter it through a 0.45μm filter. This removes impurities that could clog the filler pores and eliminates pigments that may be irreversibly adsorbed onto the filler, compromising separation efficiency.

Q10:How can I avoid air bubbles during column packing?
A10:Bubbles are prone to form when there are temperature inconsistencies. It is essential to ensure that the utensils, fillers, and buffers used are at a uniform temperature, preferably matching the ambient temperature. During column packing, use a glass rod to guide the flow of fillers, avoiding the formation of bubbles, which will effectively prevent the generation of bubbles.

Q11:How do I determine if the amount of filler is sufficient, and what specification should I choose?
A11:For ionic fillers, there is always a question of capacity, or for fillers used in tag protein purification, the choice is typically determined based on the customer's protein quantity and sample volume. For fillers serving as molecular sieves, the specification selection is generally made according to the customer's sample volume and the desired packed filler volume.

Q12:Under what circumstances should in-situ cleaning or regeneration of fillers be performed?
A12:If the same protein is purified consistently, in-situ cleaning after each elution is unnecessary. Typically, cleaning can be done after several uses. Regeneration should be considered when there is a noticeable decrease in filler capacity, a change in color, or a decrease in flow rate. When regenerating, the recommended concentration of hydrogen peroxide should not exceed 0.15M. Prolonged cleaning with high concentrations of sodium hydroxide can adversely affect the stability of the filler and potentially strip off the ligand. The use of 0.5M sodium hydroxide mentioned in the manual is intended for temporary use and not recommended for long-term application.

Q13:What is the size of the affinity chromatography column and the pore size of the sieve plate?
A13:Taking the DS0110 affinity chromatography column 12ml (1.5*8) as an example, 12ml refers to the column volume, 1.5 indicates the inner diameter of the column is 1.5cm, and 8 refers to the height of the column being 8cm. The affinity chromatography column includes an empty tube column, upper and lower caps, and two sieve plates (hydrophilic, with a pore size of 50μm).

Q14:How do I select fillers for gel filtration?
A14:1, Dextran Gel: Sephadex (In Sephadex G, a lower G value indicates higher crosslinking, resulting in lower water absorption) is suitable for desalting and buffer exchange.
2, Agarose Gel: Sepharose (offers a wide separation range).
3,Composite gel composed of agarose and glucose: Superdex, commonly known as agarose-glucose gel (high resolution, high recovery rate, short run time).

Q15:What are the principles for selecting gel filtration fillers?
A15:1,The higher the crosslinking degree of the gel, the smaller the pore size.
2,Agarose is primarily used for the separation of large molecules.
3,Dextran is often used for the separation of small molecules.
4,The coarseness of gel particles affects the separation efficiency. Our company primarily offers medium-grained products.

Q16:Please provide information on the shelf life, separation range, and maximum pressure tolerance of the Dextran Gel Series.
A16:Dextran Gel Sephadex is a commercialized gel formed through crosslinking with epichlorohydrin under alkaline conditions. It exhibits strong hydrophilicity, rapidly absorbing water and expanding in aqueous solutions. It remains stable in alkalis and weak acids and can be sterilized under high pressure. The Sephadex G series products exhibit increasing crosslinking, leading to a tighter network structure, smaller pore size, and reduced water absorption upon expansion. Consequently, they are suited for separating relatively low molecular weight substances.
Sephadex can be classified into four types based on particle size: Coarse (C), Medium (M), Fine (F), and Superfine (SF). Generally, coarse particles offer faster flow rates but poorer resolution, while fine particles, despite higher backpressure and slower flow rates, provide higher resolution. The choice of particle size depends on the separation requirements. Sephadex has relatively poor mechanical stability and is not pressure-resistant. Fine particles with high resolution require slower flow rates, limiting their use for rapid and efficient separations.
LH-type Dextran Gels, such as Sephadex G-25 and G-50, are alkylated by adding hydroxypropyl (or carboxyl) groups, resulting in Sephadex LH-20 and LH-60. These are suitable for separating lipophilic substances, such as cholesterol and fatty acid hormones, using organic solvents as the mobile phase.

Q17:What do 2B, 4B, 6B, 4FF, and HP in agarose gel represent, respectively?
A17:4B or 6B represent the content of cross-linked agarose in the agarose gel. The higher the degree of cross-linking, the smaller the pore size. Sepharose 4B has a looser structure than Sepharose 6B, yet it possesses a larger adsorption capacity compared to Sepharose 2B. Therefore, Sepharose 4B is the most widely used among them. FF stands for Fast Flow, indicating that the FF series can withstand higher flow rates, facilitating scaled-up separation and purification processes with good resolution achieved in a shorter time. HP, on the other hand, stands for High Performance, suggesting that the HP series offers even higher resolution capabilities.

Q18:What are the guidelines for selecting fillers in protein purification?
A18:The targets of downstream purification of biomolecules generally include proteins, enzymes, recombinant proteins, monoclonal antibodies, antibodies, antigens, peptides, viruses, biochemical molecules, etc. Prior to purification, it is essential to determine the various physical and chemical properties of the biomolecules, and then to select the most effective purification process through experimentation.
Measurement - Molecular Weight and PI
When the physical properties of the target protein, such as molecular weight and isoelectric point (PI), are unknown, PAGE electrophoresis or chromatography methods can be employed for determination. Fillers with a broad separation range are particularly suitable for estimating the molecular weight of unknown proteins. By using a small amount of ion-exchange media in multiple test tubes containing buffers with varying pH values, the PI can be roughly determined, and the optimal pH for the purification buffer can be selected.
Selection - Chromatographic Methods
If the characteristics of the target protein or sample composition are not well understood, several different purification methods can be attempted:
Employing the versatile gel filtration method, select a medium with a wide separation range to fractionate the sample based on molecular weight.
Use affinity chromatography media containing specific ligands or antibodies to bind the target protein. Alternatively, various activated coupling media can be used to couple substrates, receptors, or other molecules specific to the target protein, creating custom affinity media for direct binding. This approach can yield highly pure samples in a single step.
For large-volume samples, ion-exchange chromatography is often used for concentration and initial purification. Samples eluted with high salt concentrations can be further purified by hydrophobic interaction chromatography (HIC), which utilizes high-salt adsorption and low-salt elution principles. Eluted samples can then be directly loaded onto ion-exchange or other adsorptive chromatography columns. These two methods are frequently alternated in purification workflows.
Purification - Large-Scale Crude Material
When processing large volumes of crude material, to prevent column clogging, large-particle-size, high-flow-rate ion-exchange media are generally used. This approach integrates centrifugation, ultrafiltration, and initial purification, improving recovery rates and shortening purification cycles.
Purification - Ammonium Sulfate Samples
Ammonium sulfate precipitation is commonly used for initial sample purification. Treated samples under high salt conditions are well-suited for direct loading onto HIC columns. For ion-exchange chromatography, prior desalting with Sephadex G-25 is necessary. As a newer technology, HIC is increasingly integrated into various production processes due to the growing variety of media available. Samples eluted with low salt can be slightly diluted or directly loaded onto other adsorptive chromatography columns.
Purification - Carbohydrate Molecules
Immobilized lectins such as concanavalin A, peanut, and barley lectins can bind to carbohydrate residues of glycans, making them suitable for separating glycosylated membrane components, cells, and even subcellular organelles, as well as purifying glycoproteins.
For polysaccharide purification, molecular sieves like Sephadex and Sephacryl are commonly used. For molecules with a molecular weight below 600 kDa requiring higher resolution, the newer generation of Superdex can be selected. Plant materials may contain water-soluble, acid-soluble, and alkali-soluble polysaccharides. A combination of molecular sieves and ion-exchange chromatography can facilitate the further purification of individual components. Additionally, to remove proteins that may cause allergic reactions from polysaccharide drugs, traditional Sevag methods using butanol for protein removal require repeated steps. However, anion and cation exchange methods can quickly remove residual proteins from polysaccharides in one or two steps.
Purification - Glycoproteins
Lectin-coupled agarose gels, such as Con A agarose gel FF, can separate various glycoproteins. Boronate agarose gel FF is also used to separate polysaccharides and glycoproteins based on the affinity of phenylboronic acid to adjacent diols on various glycosyl residues. Purification of sugars and glycoproteins can be achieved by lowering the pH or using competitive elution methods.
Purification - Membrane Proteins
Membrane proteins exhibit relatively strong hydrophobicity and can be separated using phenyl or butyl fillers in hydrophobic chromatography. If they possess glycosylated structures or receptors, affinity chromatography can also be applied. If ready-to-use affinity fillers are unavailable, they can be synthesized using activated fillers, combined with ion-exchange and gel filtration chromatography for further purification.
Detergents are often used to maintain the activity of membrane proteins during separation. Ionic detergents should be selected with an opposite charge to the target protein to avoid competing with it for exchange media during ion-exchange chromatography, thereby facilitating detergent removal. Non-ionic detergents can be removed by hydrophobic interaction chromatography.
Purification - Monoclonal Antibodies and Antigens
Monoclonal antibodies (mAbs) are predominantly IgGs. Their sources mainly include ascites fluid and fusion tumor culture supernatants. Ascites fluid contains abundant albumin, transferrin, host antibodies, etc. Protein G and Protein A exhibit specific affinity towards the Fc region of IgGs, enabling one-step purification of IgGs from various sources (see Appendix). Recombinant Protein A exhibits higher capacity and specificity for IgGs, with reduced shedding of groups. Detached rProtein A can be readily removed using ion-exchange Q Sepharose HP gel.
Serum supplements such as calf serum can be pretreated with Protein G to remove IgGs prior to culture.
Hydrophobic interaction chromatography using Phenyl-Sepharose HP gel is also suitable for purifying IgGs, effectively removing aggregates from antibodies.
The most effective method for purifying IgG antigens involves activating coupling media, such as CNBr- or NHS-activated Sepharose gel, to conjugate with IgGs, subsequently facilitating the isolation of IgG antigens.
Pyridyl-agarose FF is suitable for the separation and purification of IgY, IgM,supercoiled DNA, among others.
Purification - Recombinant Proteins
Purification considerations should be incorporated into the design and construction of recombinant proteins. Here, we introduce three rapid expression and one-step purification fusion systems.
HIS-tagged recombinant proteins can be conveniently separated using Nickel-Sepharose FF or Nickel-NTA-Sepharose FF. These resins are pre-chelated with nickel ions, offering ease of use, higher binding capacity, and enhanced specificity. Multiple elution methods can be selected, making them optimal tools for purifying such fusion proteins. High-resolution HP-grade resins are also options.
GST fusion proteins are co-expressed with glutathione-S-transferase, facilitating separation using GST-Sepharose FF, followed by enzymatic cleavage with enterokinase or thrombin to obtain the expressed product. Thrombin can be separated using Heparin-Sepharose FF or Benzamidine-Sepharose FF.
Protein A fusion vectors enable the co-expression of the target protein with the IgG-binding domain of Protein A, facilitating purification using IgG Sepharose 6FF.
Purification - Plasma Proteins
Anion exchange chromatography is widely employed in plasma protein purification. Combined with cold ethanol precipitation, DEAE-Sepharose FF is used in the later stages of globulin purification to adsorb impurities like dimers, enhancing product purity. Similarly, anion exchange can adsorb PKA, trace IgG, and aggregates during albumin purification. In the purification of Factor VIII, large-pore Q-Sepharose gel enhances capacity and recovery. Traditional DEAE-Sephadex A-50 is also a classical method for purifying prothrombin complex concentrates (PCC).
Purification - Inclusion Body Proteins
Inclusion body proteins often need to be solubilized in 6M guanidine hydrochloride or 8M urea. The chemically stable Sepharose 6FF gel filtration medium is suitable for purification under denaturing conditions. Denatured proteins require refolding into their native conformations. Q Sepharose FF and Phenyl Sepharose FF have been found to aid in the refolding of inclusion body proteins. Generally, higher purity of inclusion body protein samples leads to better refolding outcomes.
Solid-Phase Refolding of Inclusion Body Proteins
Recent literature reports have described immobilizing (adsorbing) inclusion body proteins onto chromatography media under denaturing conditions, typically using various Sepharose FF ion-exchange media. After removing denaturants, proteins are successfully refolded on the media, followed by elution of the refolded proteins. Solid-phase refolding avoids aggregate formation during conventional refolding processes, resulting in higher refolding yields, eliminating the need for extensive sample dilution, and combining refolding with initial purification, thereby saving time and enhancing recovery rates.
Solid-phase refolding methods have also been applied to directly refold and purify histidine fusion proteins expressed as inclusion bodies using Nickel-Sepharose gel, and fusion proteins containing multiple lysines expressed as inclusion bodies using Heparin-Sepharose gel.
Purification - Peptides
Peptides originate from natural extraction, synthetic, or recombinant sources. Peptides are susceptible to enzymatic degradation but can be refolded from organic solvents or chaotropes, thus often purified using highly selective reverse-phase chromatography or ion-exchange resins.
Purification - Nucleic Acids and Viruses
Purification of Nucleic Acids is essential for eliminating contaminants that can adversely affect sequencing or PCR-based research. Nucleic acids can be broadly categorized into plasmid DNA, phage DNA, and PCR products, among others. Viruses, considered as large nucleic acid molecules, can undergo a similar purification process as plasmid DNA, involving the removal of impurities using gel filtration media such as Sephacryl S-1000 SF or Sepharose 4FF for macromolecular separation, followed by ion exchange chromatography with Q-agarose gel for nucleic acid isolation. Alternatively, enrichment and separation using DEAE or Q-agarose gel FF can precede purification on agarose gel 6B FF to obtain pure viruses.
Vaccine Purification - Viral Vaccine Purification
Gel filtration media like Sepharose 4 Fast Flow or Sepharose 6 Fast Flow represent classical approaches for purifying viral vaccines, effectively removing host cell proteins from culture media, offering high throughput and rapid processing. Columns typically range from 40-70 cm in height, with sample loading of 10-15%. Currently, this method is employed in the production of vaccines against hepatitis B, rabies, hemorrhagic fever, influenza, among others. For vaccines with smaller molecular weights, such as hepatitis A, Sephacryl S-500 HR can be utilized.
Purification - Active Ingredients from Traditional Chinese Medicines (TCMs)
The chemical composition of TCMs is exceedingly complex. Traditionally, TCMs are decocted for consumption, with most active ingredients being hydrophilic, encompassing alkaloids, flavonoids, anthraquinones, saponins, organic acids, polysaccharides, peptides, and proteins. A flexible and comprehensive application of various chromatography techniques, including ion exchange, molecular sieve, and reverse-phase chromatography, facilitates the isolation of individual active components. Dextran gel LH-20, with both adsorptive chromatography and molecular sieve capabilities, exemplifies this: in methanol-based separation of flavonoid glycosides, trisaccharides elute first, followed by disaccharides, monosaccharides, and finally aglycones. LH-20 is versatile, accommodating solvents like water, alcohols, acetone, and chloroform, and is widely used for the separation of natural products, including alkaloids, glycosides, flavonoids, quinones, lactones, terpenes, and sterols.
Alkaloids, positively charged in acidic buffers forming salts, can be separated by SP cation exchange chromatography, effectively resolving closely related structures. Conversely, flavonoids, anthraquinones, saponins, and organic acids, soluble in mildly alkaline buffers, exhibit good separation on Q anion exchange columns.
Antibiotic Polymer Analysis
Since the 2000 edition of the Chinese Pharmacopoeia, the percentage of polymers in ceftriaxone sodium antibiotics has been mandated for determination, employing the dextran gel G10 filtration method.
Removal - Nucleic Acids from Proteins
The presence of substantial amounts of nucleic acids can enhance sample viscosity, leading to zone broadening, increased backpressure, and subsequent reductions in resolution and flow rate. Regulatory agencies for pharmaceuticals and food safety impose strict limits on nucleic acid content. The issue is particularly acute with intracellularly expressed proteins. Nucleic acids, being negatively charged, can be efficiently removed during initial purification stages by utilizing cation exchange media such as SP or CM Sepharose FF, which bind the target proteins while excluding nucleic acids. Nucleic acids dissociate from proteins in high salt conditions, rendering hydrophobic chromatography media suitable for simultaneous protein purification and nucleic acid removal. Furthermore, nucleases can be employed to fragment nucleic acids into smaller pieces, facilitating their removal during subsequent gel filtration-based purification steps.
Removal - Viruses and Microorganisms
Viruses and microorganisms pose pathogenic risks and should be minimized. Integration of various chromatography techniques, the use of water for injection, and regular in-place sanitization and cleaning of instruments and gels with NaOH can effectively prevent the accumulation of contaminants.
Most viruses possess lipid envelopes, which can be targeted for inactivation using S/D (solvent/detergent) treatments with opposite charges to the target protein, such as Triton and Tween. Following inactivation, appropriate ion exchange media like CM Sepharose FF can be employed to bind the target protein while removing S/D agents.
Removal - Serum Albumin
Blue agarose gel FF is a valuable tool for separating various kinases or enzymes dependent on nucleotides as coenzymes. Its specificity for binding albumin enables the removal of serum albumin interference from samples, enhancing sample purification. At a pH of approximately 4.5, 1 ml of the resin can remove albumin from 0.3 ml of plasma with a loading capacity of 15 mg/ml. Nickel agarose gel FF also exhibits albumin-binding capability, with a loading capacity ranging from 5-20 mg/ml.
Desalting and Removal of Small Molecules
Gel filtration media like Sephadex G10, G15, and G25 efficiently remove small molecules, processing up to 30% of the bed volume. By collecting the first 1/3 to 1/2 column volumes of eluate after sample application, molecules smaller than the upper separation limit of the media can be readily removed, making the process straightforward. Since only small molecules are being removed, a column height of over 10 cm suffices. The entire process typically takes only a few minutes to half an hour. The Sephadex G25 series is specifically designed for protein desalting.
For viruses, DNA vaccines, or plasmids, agarose gel 6B/FF can be used for desalting or buffer exchange. Proteins and antibodies, on the other hand, can be rapidly desalted and buffer-exchanged using agarose gel G15/FF or dextran gel G25. The former is more suitable for molecules larger than 3000 Da, offering a broader application range compared to conventional Sephadex G25.

Q19:What are the strong anion exchange chromatography media (QAE, Q)?
A19:S8851 Q-Agarose Gel FF
S9200 Q-Agarose Gel HP
S9820 QAE-Dextran Gel A-25
S9830 QAE-Dextran Gel A-50

Q20:What are the strong cation exchange chromatography media (SP, S)?
A20:S8861 SP-Agarose Gel FF
S9220 SP-Agarose Gel H.P.
S9800 SP-Dextran Gel C-25
S9810 SP-Dextran Gel C-50

Q21:What are the weak anion exchange chromatography media (DEAE)?
A21:S8800 DEAE-Agarose Gel FF (GE Packaged)
S8801 DEAE-Agarose Gel FF
S8791 DEAE-Agarose Gel CL-6B
D8300 DEAE-Dextran Gel A-25
S9130 DEAE-Dextran Gel A-50
D8900 DEAE-Cellulose DE-32
C8930 DEAE-Cellulose DE-52
C8350 DEAE-Cellulose DE-52 (Imported)

Q22:What are the weak cation exchange chromatography media (CM)?
A22:S8781 CM-Agarose Gel FF
S9230 CM-Agarose Gel CL-6B
S8240 CM Sephadex C-25 (CM-Dextran Gel C-25)
S8180 CM-Dextran Gel C-50
C8691 Carboxymethyl Cellulose CM-52

Q23:What are the types of media used for gel filtration chromatography?
A23:Dextran Gels: Sephadex (the smaller the G value in Sephadex G, the higher the cross-linking degree and the lower the water absorption) suitable for desalting and buffer exchange.
Agarose Gels: Sepharose (with a broad separation range).
Superdex Gels: A composite gel of agarose and glucose, featuring high resolution, high recovery rates, and short run times.
Polyamide Gels: Higher mesh numbers lead to better separation efficiency and a wider range of separable substances, but more complex separation conditions must be explored. Mesh sizes of 60-100 are commonly used for rough separations, while 200-300 are used for fine separations, in conjunction with the complexity of the elution system and separation system.

Q24:What is the series of agarose gel products?
A24:Agarose is a polysaccharide chain composed of D-galactose and 3,6-anhydro-L-galactose linked together. It is liquid at 100°C and spontaneously aggregates into beads to form a gel through intermolecular hydrogen bonds when cooled below 45°C. However, it has poor stability. Agarose molecules interconnect to form linear double-stranded single-ring agarose, which further aggregates into agarose gel.
Agarose gels are prone to rupture when stored in a dry state, thus they are typically preserved in solutions containing preservatives. Aging occurs easily above 40°C. High pressure and freezing are not recommended.
Commonly used products include Sepharose 2B, 4B, and 6B. Sepharose 4B is the most widely applied due to its looser structure compared to Sepharose 6B and its higher adsorption capacity compared to Sepharose 2B.
Sepharose CL is generated by reacting with 1,3-dibromo-2-propanol under strong alkaline conditions, resulting in CL-type cross-linked agarose. This improves both thermal and chemical stability, making it especially suitable for separations involving organic solvents and capable of enduring rigorous in-place cleaning.
The FF series can withstand higher flow rates, facilitating scaled-up separation and purification with good resolution achieved in a short time.