Bio-Rad EXQuest Spot Cutter - Manual (Page 218)

Analysis Tools
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The raw quantity of each spot in a gel will be divided by the total quantity of the spots
in the analysis set in that gel, according to the normalization formula.
Note: The advantage of selecting multiple housekeeping proteins is that occasional
minor variations in the size and/or intensity of a particular spot will be averaged
out when the group of spots is considered is a whole.
Total Quantity in Valid Spots
In this normalization method, the raw quantity of each spot in a member gel is divided
by the total quantity of all the spots in that gel that have been included in the Master
(see the normalization formula).
Select the Total Quantity in Valid Spots option button in the Normalization dialog to
activate this method. This normalization method assumes that few protein spots
change within the experiment, and that the changes average out across the whole gel.
It can be useful if very little information is known about the possible source of sample
variation.
Total Density in Gel Image
In this method, the raw quantity of each spot in a member gel is divided by the total
intensity value of all the pixels in the image (see the normalization formula).
Select the Total Density in Gel Image option button to activate this normalization
method. This model assumes that the total density of an image (i.e., background
density plus spot density) will be relatively consistent from gel to gel. It can be useful
if very little information is known about the possible source of sample variation.
Specified Value
In this method, the raw quantity of each spot in a member gel is divided by a user-
defined normalization factor for that gel.
Select the Specified Value option and enter a normalization value for each gel in the
table at the bottom of the dialog box. This value will be used as the denominator in
the normalization formula.