Hi I have this antibody that I need to run on gel. But it’s been purified under high salt conditions (0.5M NaCl). I know that the gel will give me problems if I run it under the high salt. However, I have been told I can microdialyze my protein. but how??
Have you considered using a quick-spin column to exchange your buffer?
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Hi Helix
Welcome to the community. Quick Spin columns are certainly good options for desalting the samples. There is one caveat though and that is with desalting columns you can lose Low Molecular weight Globular Proteins (below 30KDa).
Have you noticed the same?
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Sachin
Use dialyzing membranes with MWCO 500-1000Da. Dialyze your protein sample against a low salt concentration. If you intend to lyophilize this protein, then use a volatile buffer such as Ammonium Bicarbonate (50mM should work well). If you dont intend to lyophilize, then the same buffer works well with one and two dimensional electrophoresis.
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ARGERINE
Don’t you think that sample will get diluted after dialysis. I mean e.g. if i start with 10mg/ml protein conc. and my dialysis membrane has 200microlitre sample. Wont it swell up in volume upon dialysis? This is a major reason why my former boss was never in favor of dialyzing membranes but advocated using Centrifugal filters.
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That shouldn’t be a concern Navneet. You can easily concentrate your sample prior to elution from dialyzing membranes and in fact even use the membranes to concentrate your sample. A simple trick we use is after dialysis, put your sample around sucrose powder and it will become concentrated to the volume you require.
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That is the easiest way to concentrate a sample and in quick succession. It’s also important to note that this method is not dependent upon any of the special equipment requirements like freeze dryer or rotary Vac systems.
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