Phage concentration

The phage concentration is simply obtained by measuring the absorbance at 265 nm... 

In this strategy, it is very important to avoid interphage catalysis. Therefore, the phage concentration should be kept low (lower than 10-9 M) and phages should not be precipitated with PEG. Because removal of excess label is generally required before affinity capture, it should be done by phage dialysis or size-exclusion chromatography. 

When displaying an enzyme on a phage surface, evaluation of the level of functional enzymes attached to the phage particle is generally recommended prior to mutagenesis and selection. This level is equal to the ratio between the enzyme and the phage concentrations. 

The presence of the enzyme-g3p fusion protein should also be detected by western blot analysis with an anti-g3P and/or an anti-enzyme antibody. With anti-g3p detection, the relative intensities of the fusion and the free g3p proteins should be in qualitative agreement with the displayed level evaluated by the ratio between the enzyme and the phage concentration. 

Determine the phage concentration spectrophotometrically (OD268 = 1.0 for a solution of 5 x 1012 phage/mL). 

Fig. 3. Spectral scan of phage solution. Quantification of the physical units (virions/ mL) of a phage solution is achieved by performing a spectral scan of a dilute phage solution. The protein to DNA weight ratio for filamentous phage is about 6 1. Thus the UV absorption spectrum can be utilized for the calculation of phage concentration in virions/mL (8).

Since the initial phage concentration may vary from as little as 1 mg. (220) to 100 mg. per liter, concentration is required either as a separate or... 

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