I have used phosphate buffer to dilute the pellet of enzyme. The enzyme became so much diluted so kindly tell me how to concentrate the enzyme within one day?
Use concentrators, and as long as the protein get concentrated, add very diluted buffer and reconcentrate, in order to avoid phosphate hyperconcentration and possible precipitation of crystals.
Use a centricon (There are many options available in the market with different kDa cut/offs Eg.Millipore), You can also try methods such as ion exchange chromatogrpahy (use low elution volumes) or ammonium suplhate precipitation.
These types of units can be used:
http://www.emdmillipore.com/US/en/product/Amicon-Ultra-15-Centrifugal-Filter-Units,MM_NF-C7715
They are available in a range of molecular-weight cut-off (MWCOs) filters, and are also available in 500 µL (max.) volume. Each unit takes around 10 minutes to spin down.
Ross
The best method is the diafiltration to eliminate salys followed by a concentration using a filtration membrane (Amicon cell or other) with a low molecular weight 3,5 or 10 kDa.
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