【资源】蛋白质浓缩，两个小tips
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1. 如何选用透析/超滤膜？ （这个问题很多人问过:D）
Dialysis membranes are available in a number of thicknesses and pore sizes. Thicker membranes are tougher, but restrict solute flow and reach equilibrium more slowly. Pore size is defined by “molecular weight cutoff” (MWCO)—i.e., the size of the smallest particle that cannot penetrate the membrane. The MWCO designation should be used only as a very rough guide; if accurate MWCO information is required, it should be determined empirically. See Craig, 1967, for a discussion of parameters affecting MWCO. Knowledge of the precise MWCO is usually not required; however, it is necessary only to use a membrane with a pore size that is much smaller than the macromolecule of interest. For most plasmid and protein dialyses, a MWCO of 12,000 to 14,000 is appropriate, whereas a MWCO of 3500, 2000, or even 1000 is appropriate for peptides.
Most dialysis membranes are made of derivatives of cellulose. They come in a wide variety of MWCO values, ranging from 500 to 500,000, and also vary in cleanliness, sterility, and cost. Spectrum has the most impressive inventory. The least expensive membranes come dry on rolls; these contain glycerol to keep them flexible as well as residual sulfides and traces of heavy metals from their manufacture. If glycerol, sulfur compounds, or small amounts of heavy metals are problematic, cleaner membranes should be purchased or membranes should be prepared as described below. Protein dialysis should only be done with clean membranes.
It should be noted that several commercial kits for performing microdialysis are also available. For example, use of microdialysis cassettes (Slide-A-Lyzer, Pierce or Dispo-Dialyzer, Spectrum) eliminates the need for boiling or presoaking dialysis tubing as well as the cumbersome task of clamping the dialysis tubing. Other vendors provide vessels for dialyzing multiple samples simultaneously. Selection of the best dialysis approach to be used will depend on a number of factors including the volumes to be dialyzed, the MWCO appropriate for the sample, and, of course, costs when considering the use of kits.
2. 这些浓缩方法你知道吗？
Because it is usually more convenient to store antibodies or other proteins at >1 mg/ml, it may be necessary to concentrate the solution. In addition, concentration of protein solutions is often necessary to decrease the volume of a sample; e.g., to load onto a size-exclusion column when the volume must be £5 ml.
There are several simple and relatively inexpensive methods for concentrating protein solutions. Dialysis against Aquacide 11A (Calbiochem), which removes water through the dialysis tubing, may be used. After concentration, the solution must be redialyzed into the appropriate buffer. Another method is to use Immersible-CX Ultrafilters (Millipore) which, when connected to a vacuum, remove everything below their molecular weight cutoff (MWCO). Alternatively, Centricon microconcentrators (Amicon), which are available in different MWCO sizes, are very useful for concentrating small amounts of material in small volumes. Follow the instructions from the manufacturers when using these products.
Dialysis membranes are available in a number of thicknesses and pore sizes. Thicker membranes are tougher, but restrict solute flow and reach equilibrium more slowly. Pore size is defined by “molecular weight cutoff” (MWCO)—i.e., the size of the smallest particle that cannot penetrate the membrane. The MWCO designation should be used only as a very rough guide; if accurate MWCO information is required, it should be determined empirically. See Craig, 1967, for a discussion of parameters affecting MWCO. Knowledge of the precise MWCO is usually not required; however, it is necessary only to use a membrane with a pore size that is much smaller than the macromolecule of interest. For most plasmid and protein dialyses, a MWCO of 12,000 to 14,000 is appropriate, whereas a MWCO of 3500, 2000, or even 1000 is appropriate for peptides.
Most dialysis membranes are made of derivatives of cellulose. They come in a wide variety of MWCO values, ranging from 500 to 500,000, and also vary in cleanliness, sterility, and cost. Spectrum has the most impressive inventory. The least expensive membranes come dry on rolls; these contain glycerol to keep them flexible as well as residual sulfides and traces of heavy metals from their manufacture. If glycerol, sulfur compounds, or small amounts of heavy metals are problematic, cleaner membranes should be purchased or membranes should be prepared as described below. Protein dialysis should only be done with clean membranes.
It should be noted that several commercial kits for performing microdialysis are also available. For example, use of microdialysis cassettes (Slide-A-Lyzer, Pierce or Dispo-Dialyzer, Spectrum) eliminates the need for boiling or presoaking dialysis tubing as well as the cumbersome task of clamping the dialysis tubing. Other vendors provide vessels for dialyzing multiple samples simultaneously. Selection of the best dialysis approach to be used will depend on a number of factors including the volumes to be dialyzed, the MWCO appropriate for the sample, and, of course, costs when considering the use of kits.
2. 这些浓缩方法你知道吗？
Because it is usually more convenient to store antibodies or other proteins at >1 mg/ml, it may be necessary to concentrate the solution. In addition, concentration of protein solutions is often necessary to decrease the volume of a sample; e.g., to load onto a size-exclusion column when the volume must be £5 ml.
There are several simple and relatively inexpensive methods for concentrating protein solutions. Dialysis against Aquacide 11A (Calbiochem), which removes water through the dialysis tubing, may be used. After concentration, the solution must be redialyzed into the appropriate buffer. Another method is to use Immersible-CX Ultrafilters (Millipore) which, when connected to a vacuum, remove everything below their molecular weight cutoff (MWCO). Alternatively, Centricon microconcentrators (Amicon), which are available in different MWCO sizes, are very useful for concentrating small amounts of material in small volumes. Follow the instructions from the manufacturers when using these products.
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