Weight Exclusion Column Chromatography

[Hypercube]

We recently did a lab involving a mixture of three proteins; two known and one unknown, and the goal is to determine the identity of the unknown. After we took the absorbances of all the fractions at three different wavelengths, we were asked to "prepare a graphof OD600 that includes all three absorbance profiles". I have absolutely no idea what OD600 is, since we did not take the absorbance at 600 nm, nor how I could put all three wavelengths on one graph.

 

Any help would be greatly appreciated.

[MedGen]

We recently did a lab involving a mixture of three proteins; two known and one unknown, and the goal is to determine the identity of the unknown. After we took the absorbances of all the fractions at three different wavelengths, we were asked to "prepare a graphof OD600 that includes all three absorbance profiles". I have absolutely no idea what OD600 is, since we did not take the absorbance at 600 nm, nor how I could put all three wavelengths on one graph.

 

Any help would be greatly appreciated.

 

OD is optical density which refers to the absorbance per unit of length, which in most cases is 1, i.e. the distance through which the light has to pass through the sample. If you are using a typical 1cm cuvette then it is equal to the absorbance.

 

It is possible to put all three curves on one graph with Excel, I think you have to select a line graph and put them in as 3 separate samples (in the X variable box). The only problem is that you don't get a smooth curve, which sucks. Alternatively draw it by hand.

[CharonY]

At which wavelength did you do the measurements?

[nandini]

hi,

it would be nice to know at what different wavelength you have taken your absorbance. for proteins absorbance is usually measured at 280nm ( which is the absorbance maxima for the aromatic side chains ) so depending on the number of aromatic rings present in the protein you will get the absorbance values. OD 600 in when you measure the absorbance at 600nm wavelength, this is usually the absorbance maxima for nucleic acids and by checking the OD at 600nm you know how much nucleic acid contamination you have in your protein solution.