Bio-Rad EXQuest Spot Cutter - Manual (Page 216)

Analysis Tools
6-25
Pipetting error compensation is only available if you have calibrated the gels using
Calstrips and entered the number of counts loaded in the gel into the Gel Record. See
Appendix J.
After you have selected the settings described below, click Done.
6.7.a Scaling Factor
The scaling factor is a constant by which the normalized quantity is multiplied to give
a more meaningful value. A common scaling factor is 10
6
, giving units of parts per
million (PPM).
Under Scaling, specify the scaling factor that you would like to use by clicking on
PPM (x 1000000), Percent (x 100), or User Specified. If you select User Specified,
enter your own scaling factor and units in the appropriate fields.
6.7.b Normalization Methods
The normalization method you select will depend in part on how much you know
about your gels and how they were prepared. In the Normalization dialog box, select
from the options for your Basis or denominator. For example, if you can readily
identify housekeeping proteins on your gels, the Total quantity in analysis set method
is preferable. If you know that each gel was loaded with a different amount of sample,
you can select Specified Value and enter the known variation factor.
Or, check the Total quantity in analysis set option and select your set of housekeeping
proteins from the pulldown list.
Total Quantity in Analysis Set
This normalization method involves identifying a group of protein spots that are
present in all MatchSet members and are conserved (i.e., their quantities should not
vary between samples). These are proteins that you know are expressed in a very
consistent manner at all times within a cell, with little or no regulation. They are
commonly referred to as housekeeping proteins.
Step 1: Create an arbitrary analysis set containing several of these housekeeping proteins
from different regions of the MatchSet Master. To create an arbitrary analysis set,
select Analysis Set Manager... from the Analyze menu.