Unlike spherical phage, such as T4 and λ, which have roughly equal weight ratios of protein to DNA, filamentous phage have about six times more protein than DNA; the protein therefore contributes substantially to the absorption spectrum. Based on the measurements of Day and Wiseman, the concentration of phage in virions/ml is as seen. Subtracting the A320, a wavelength where there is little light absorption from phage chromophores, is meant to correct crudely for light scattering from phage particles and non-phage particle contaminants.
Protocol
1. Prepare a serial dilution (in 1X TBS) of the filamentous phage to be analyzed if there is evidence that the solution is too concentrated for absorption analysis.
2. Make sure the exterior surfaces of two quartz cuvettes are clean (especially the window through which the light will travel, see Tip #1).
3. Turn on the spectrophotometer and allow the instrument to warm up for an appropriate period of time (refer to the Manufacturer's instructions).
4. Program the spectrophotometer to analyze a spectrum from 240 nm to 320 nm and to also report the absorbance value at 269 nm and 320 nm.
5. Add 250 μl to 1 ml of 1X TBS (depending on cuvette) to the cleaned cuvettes and place one of the cuvettes into the reference cell and the other into the analysis cell.
6. Blank the spectrophotometer.
7. Remove the cuvette from the analysis cell and aspirate the 1X TBS.
8. Add one of the solutions to be analyzed into the cuvette.
9. Place the cuvette in the analysis cell and determine the spectrophotometer spectrum (see Tip #2).
10. Repeat Steps #7 to #9 for the remaining samples.
Buffers
TBS (10X)
Store at room temperature
500 mM Tris-HCl pH 7.5
1.5 M NaCl
Materials
Tris-HCl
Sodium Chloride
Method Tips
1. Clean two quartz cuvettes by washing the exterior of the cuvette with 100% Methanol.
2. A typical UV absorption spectrum of purified filamentous phage dissolved in TBS results in a broad plateau at 260 to 280 nm, with a shallow maximum at 269 nm
References
Day, L.A. and Wiseman, R.L. A comparison of DNA packaging in the virions of fd, Xf, and Pf1. In: The Single-Stranded DNA Phages. (1978) (Denhardt, D.T., Dressler, D. and Ray, D.S. ed) p. 605-625.
