Alex,
I do not recall any specific explanation of the numbers usually given as a
2D gel separating power, but I do believe that these numbers are based on
geometry rather than protein chemistry. With the assumption that an
average gel is 16x16 cm in size and an average spot is between 2 and 3 mm
in diameter, there is room for 2500 - 6400 spots on a gel. This assuming
perfect separation and ideal distribution of spots on a gel which - of
course - does not happen. Average gel run with the use of carrier
ampholytes pH 3.0 - 10.0 will produce pH gradient from approx 3.0 to approx
7.5. Molecular mass range will depend on a second dimension gel acrylamide
concentration. This will leave some of the whole cell extract proteins
"outside" the gel. And since the distribution of spots on a gel will
reflect the pI and molecular mass distribution of the whole cell extract
proteins, this leaves us with approximately only 50% of the gel area with
the highest concentration of spots. So based on all these assumptions I
would estimate separation power of 16x16 cm 2D gel as about 1000 - 2500
spots per gel. In my practice I was able to see 1000 - 1500 spots (with
silver staining).
In terms of a peak (spot) capacity I was able to see good separations
loading 500 ug total protein from the whole cell extract. This gives
approx 0.25 - 0.5 ug per spot. The most intense spots such as actin are
probably about 5 ug, the weakest spots at the silver staining level contain
probably about 0.01 ug. All the above is only an approximation. Results
may vary a lot depending on the carrier ampholytes used (or IPG), pH (pI)
range, acrylamide concentration, sample preparation and experience level of
an operator.
I hope this will be sufficient as a general idea. And I hope that 2D gel
gurus will correct my possible "math" errors.
Jacek Mozdzanowski
Bioanalytical Sciences
SmithKline Beecham