I would say it really depends on what you are aiming at when making the lysate. In general 300 mM is about double ionic strength and osmolarity that nature provides the cells with (which is 140 mM), keeping in mind that inside cells there is actually KCl. So higher osmolarity will help lysing cells (by implosion), higher ionic strength as already stated will help breaking down protein complexes and interactions and solubilizing proteins. In these conditions pull-downs and IPs are quite stringent, so weaker interactions will be lost and only tight ones will be detected. Might be good for studying certain sticky proteins.
As viviana stated thats true..NaCl helps to mimic the physiological condition..it also gives ionic strength during the purification with IMAC (NI-NTA) matrix and sometimes helps in binding with the NI-NTA..if your protein does not binding or less binding with the matrix, we prefer high concentration of NaCl as well...after purify the protein its better to dialysis the protein with lower concentration of NaCl such as 150mM...
Despite mimicking the physiological condition, NaCl can basically help to increase the ionic strength of the native or denaturing purification buffers. It helps to remove the contaminants from the Ni-NTA resin by reducing the non-specific hydrophobic and ionic interaction binding between the protein. Usually up to 2M NaCl can be used in the buffers. In my experience, it provides better washing and elution of the his-tagged protein with higher purity.
You will have more proteins in the solution by using a high NaCl concernation buffer after a spin.
I would say it really depends on what you are aiming at when making the lysate. In general 300 mM is about double ionic strength and osmolarity that nature provides the cells with (which is 140 mM), keeping in mind that inside cells there is actually KCl. So higher osmolarity will help lysing cells (by implosion), higher ionic strength as already stated will help breaking down protein complexes and interactions and solubilizing proteins. In these conditions pull-downs and IPs are quite stringent, so weaker interactions will be lost and only tight ones will be detected. Might be good for studying certain sticky proteins.
Many buffers contain NaCl to help keep proteins soluble and to mimic physiological conditions. In chromatographic separations, like gel filtration and Ni2+ affinity columns, you may want to increase the salt concentration even more. I’ve gone up to 500 mM NaCl to give ionic strength which helps prevent nonspecific interactions between proteins and the column
Hi Stephania,
high salt in samples run on standard Tris/Glycine denaturing gels has several effects. It typically creates artifacts in the run, it has been shown to affect the interaction of the proteins with SDS, which is the driving motor in the run of the proteins, and in general, a bit counter-intuitively, larger proteins are slowed down more than smaller proteins. But you can end up with lateral diffusion, deformed band shapes, etc... all affecting your resolution. Would probably be best to precipitate your protein samples in acetone (add 10 vol. of pre-cooled acetone at -20 °C and leave O/N at -20 °C) or with TCA, if you are experienced with that. Of course you should keep in mind that after this type of protein precipitation some proteins aggregate and are lost in an insoluble pellet, even in Laemmli with boiling (if your protein of interest falls in this category you can also try re-solubilizing in 8M urea with or without thiourea). Other possibility would be a desalting using a Centricon-type cartridge: it will of course concentrate the proteins, but you can then dilute them directly in Laemmli buffer... should be quicker than a dialysis which on an extract would probably produce quite some degradation, even with protease inhibitors.
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