Biosynthetic labeling

How long should cells be labeled?

The ideal length of time to label cells depends on the protein of interest and the label that you are using.If you want to label an unstable protein with ³⁵S-methionine,a short labeling interval--no more than 2 hr--is best.If you are studying a stable protein,a longer label may be preferable.The issue is the half-life of the protein of interest relative to the half-life of the background bands.The half-life of total cellular protein is 45 to 50 hr.

Labeling with ³²Pi is different.

In most cases,the phosphate in proteins undergoes continual turn-over.Therefore,both old and newly-synthesized proteins become labeled soon after ³²Pi is added to the cells.A short labeling period with ³²Pi is advantageous in that the labeling of RNA and DNA is less obvious than in cells labeled over-night.Additionally,for those of you studying tyrosine protein kinases,phosphotyrosines tend to turn-over faster than the bulk of either phosphoserine or phosphothreonine and thus are preferentially labeled during brief labeling.

If,however,you want to look at the steady-state abundance of phosphate in proteins,lipids,or RNA,an over-night labeling period may be the best.Under these conditions,you can be reasonably certain that the specific activity of the ATP pool in the cell and of the phosphates in macromolecules is beginning to approach that of the medium.Additionally,the amount of label present in proteins,lipids and RNA should now reflect the amount of phosphate present,rather than the rate of turn-over of the phosphate.

An issue to consider however is the fact that radiation damage can induce the stabilization of p53 and cause cell cycle arrest.Cells labeled for a prolonged period with ³²P may therefore not be growing when you harvest them.

Many years ago,Mike Weber and Doc Edlin concluded that the specific activity of the ATP pool had come to equilibrium with the phosphate in the medium by 6 hours.There may therefore be no reason to label for longer with ³²Pi.

Overnight labeling is best done in medium containing a reduced concentration of phosphate or methionine.10% is often reasonable,depending on the cell line.This is easily accomplished by using methionine-free or phosphate-free medium and 10% undialyzed serum,which can be assumed to contain the same concentration of methionine or phosphate as normal medium.

For short termlabeling--30 seconds to 5 hrs--

use (1)medium completely lacking either phosphate or methionine,

(2)serum which has been dialyzed against saline,and

(3)a fairly low volume of medium;

0.75 ml for a 35 mm dish,2 to 2.5 ml for a 50 mm dish,and 2.5 to 5 ml for a 100 mm dish.

It is a good idea to rinse the cells you are going to label with labeling medium--which lacks label--prior to adding the actual labeling medium.Starvation doesn't help much.