I want to resuspend a lyophilized protein in buffers that mimick these cellular compartments. Do you know any references about that? I guess I should use a crowding agent but it should be nonproteinaceous.
5/1 medium has 85 mM KCl, 17 mM NaCl, 1 mM MgCl2, 0.2 mM CaCl2, 2 mM DTT, 1 mM PMSF, and 10 mM Tris-HCl or HEPES-KOH (the difference is important mostly for ion exchange chromatography, the buffering ion should not bind to the column). I have used it to isolate cytosolic proteins and measure their activities. The name refers to the K/Na and Mg/Ca ratios. The DTT maintains a strong reducing potential to protect -SH groups in proteins. Note that PMSF in water has a half-life of 20', add it immediately before use from a 1 M stock in i-propanol (not DMSO, which makes skin permeable to the highly toxic PMSF!). The ionic strength is ~230 mOsm, you can add sucrose or glycerol if you want it higher, these osmolytes also have a protective effect on proteins. Other additions like nucleotides may be required to keep "your" protein happy.
The nuclear pore is selective mostly for large molecules (> 10 kDa), thus, this buffer should also mimic nucleoplasm reasonably well.
5/1 medium has 85 mM KCl, 17 mM NaCl, 1 mM MgCl2, 0.2 mM CaCl2, 2 mM DTT, 1 mM PMSF, and 10 mM Tris-HCl or HEPES-KOH (the difference is important mostly for ion exchange chromatography, the buffering ion should not bind to the column). I have used it to isolate cytosolic proteins and measure their activities. The name refers to the K/Na and Mg/Ca ratios. The DTT maintains a strong reducing potential to protect -SH groups in proteins. Note that PMSF in water has a half-life of 20', add it immediately before use from a 1 M stock in i-propanol (not DMSO, which makes skin permeable to the highly toxic PMSF!). The ionic strength is ~230 mOsm, you can add sucrose or glycerol if you want it higher, these osmolytes also have a protective effect on proteins. Other additions like nucleotides may be required to keep "your" protein happy.
The nuclear pore is selective mostly for large molecules (> 10 kDa), thus, this buffer should also mimic nucleoplasm reasonably well.
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