Contracted services
- Is it possible to manufacture beads with particle sizes other than those in the lineup?
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We do not manufacture beads with particle sizes other than those in our lineup.
- What is the general process used to bind ligands to the beads?
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When binding ligands to NHS beads, for the binding reaction, 4 concentrations are prepared (ligand solutions: 0, 0.1, 0.3, 1 mM) in order to determine the optimal concentration. The amount of bound ligands depends on the ligand properties, and varies approximately from several nmol to 100 nmol. An amount of several nmol to several tens of nmol is best for achieving purification results. Too much ligand can cause non-specific adsorption and steric hindrance, which adversely affects affinity purification.
- What amount of the beads is required?
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20 mg of beads are sufficient for 2 studies of the affinity purification conditions. A total of approximately 50 mg of beads are necessary in order to secure a certain amount of the purified target proteins or other substance after the study of the conditions.
- What are the important points when designing a ligand?
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When designing a ligand in order to introduce a functional group for the parent compound of the protein you seek to identify, because the protein which will be purified varies depending on the location where the function group is introduced, it is recommended that receptor identification tests be performed by binding multiple ligands at different linker binding locations on the same parent compound. In the event that there is no functional group capable of binding to the ligand, it is necessary to change the chemical structure of the compound itself.
- Is it possible to bind secondary amines?
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Secondary amines can be bound to NHS beads. However when both primary amine and secondary amine are present, the reaction will occur selectively with the primary amine.
- Are there any methods other than HPLC for verifying whether or not ligand binding has been successful?
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The proteins can be actually bound to the ligands in order to verify binding. At a binding concentration of 0 mM, there is no protein binding. If the protein band increases as the binding concentration increases, then good binding has been verified.
- What is the purification efficiency?
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When ligands with a relatively strong bonding strength are used, the results of studies by our company show a purification efficiency of approximately 50%. However the actual efficiency is highly dependent on the properties of the individual ligands and varies on a case-by-case basis.
- What is the optimal bead type for binding proteins?
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NHS beads are best. It is immobilized by the ε-NH2 groups in the lysine residues of the protein and the NHS groups on the bead surface. In the case of His-tag protein, immobilization on Ts beads is possible.See below for details.
https://fgb.tamagawa-seiki.com/english/selection/immunoprecipitation
- What is the efficiency when binding proteins?
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NHS beads are best. It is immobilized by the ε-NH2 group in the lysine residue of the peptide and the NHS group on the bead surface.
- How is the cell extract prepared?
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We recommend the Dignam method. However, since the Dignam method requires a large amount of cells, we recommend a method of solubilizing with a surfactant such as NP-40 when using a small amount. Please refer to the protocols 401 and 402 from the protocol page. If the above is difficult, you can also use commercially available kits (ProteoExtract Subcellular Proteome Extraction Kit (MERCK MILLIPORE), CelLytic M (SIGMA), etc.). However, if the detergent concentration is 1% or higher, it may prevent the ligand from binding to the target protein during affinity purification, so reduce the detergent concentration to 0.1% by dialysis or dilution before use.
- Is there any problem with using frozen stock homogenate?
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There is no problem. However perform centrifuge separation before using to remove any impurities (such as degenerated proteins).
- How much protein supply is necessary?
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Serum proteins or extracts from cultivated cells or tissues can be used. When using cultivated cells as the protein source, 109 nuclear proteins or 107 – 109 cellular proteins are required.
- What should be done when a large number of bound protein bands are detected?
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Change the buffer composition and detect a more highly specific band. Alternatively, it is necessary to perform competitive inhibition tests and drug elution, and verify whether or not the band is specific to that ligand. In addition, the use of active or inactive ligand-immobilized beads makes it easier to narrow down the target protein.
- Is it necessary to use the recommended buffer as the binding buffer?
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There are no problems with using TBS in place of HEPES, and NaCl in place of KCl.
- Why is it that both salt elution and boil elution are performed for elution?
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This is because bonds are divided between somewhat weaker bonds (which can be broken by salt) and strong bonds (which can be broken by a sample buffer plus heating). However there is no problem with performing boil elution alone.
- Does it happen that the band of bound protein becomes thin when the concentration of ligand is increased?
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When the amount of ligand immobilization is increased, the beads become hydrophobic and easily aggregate, which may reduce the reaction efficiency with the protein and thin the band. If bead agglomeration is seen, reduce the amount of ligand immobilization.
- Why can’t I see any bands of bound proteins?
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Since the amount of ligand immobilization may be small, consider increasing the immobilization concentration. The affinity between the ligand and the target protein may be weak, so try lowering the salt concentration in the buffer.
Also, if the target protein is low in the protein solution, you may need to consider increasing the concentration or volume of the protein solution.
- Is the optimal binding reaction time of 4 hours?
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In our experience, 4 hours is sufficient, but it is advisable to investigate the optimal reaction time as it depends on the nature of the ligand immobilized on the beads and the target protein.
- What is the optimal bead type for immobiliding antibodies?
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The optimum beads vary depending on the method of use and the bonding method. See here for details.
- Can I quantify the immobilization amount of the antibodies on the beads?
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It can be carried out by direct quantification of the immobilized antibodies. Please refer to Protocol 107 on our website.
- What is the efficiency when immobiliding antibodies?
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If binding is performed when 50 μg of antibodies is supplied to 1 mg of NHS beads, approximately 20 – 40 μg will be bound, although the result varies depending on the antibody animal, subclass, and clone. It is possible to increase the amount that is bound by increasing the amount on feed. When streptavidin beads are used, up to approximately 10 μg of biotin-modified antibodies will bond to 1 mg of Streptavidin beads or NeutrAvidin beads, although this also varies depending on the antibody.
- What is the optimal bead type for binding DNA?
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Single-stranded and double-stranded DNA can be immobilized to the Plain beads.
- What method is used to bind RNA？
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Either bind synthesized RNA with a suitable functional group (NH2 radical, SH radical, etc.) introduced onto the ends, or by binding DNA in advance and using hybridization to bind the RNA.
- I want to analyze bound proteins with MS, but what should I do if the target protein band is thin?
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Increase the scale of affinity purification (ex: 2.5 mg / 1000 μL), or increase the number of samples under the same conditions to perform affinity purification.
- How much protein can be analyzed by MS?
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It depends on the type of equipment used, but it can be analyzed if the amount of protein is about 50 ng.
