CoV2Research - May2020 - 23

2D-DIGE
The starting CHO Harvest sample and the CHO AAE
eluate were each labeled with Cy3 and with Cy5 respectively. The labeled samples were mixed according to the table below and separated by 2D electrophoresis on large format gels.
Gel ID #

LF 855 #6
LF 855 #7

Sample

μl loaded

μg loaded

CyDye

CHO Harvest

250

Cy3

CHO AAE Eluate 75

250

Cy5

CHO AAE Eluate 75

250

Cy3

CHO Harvest

250

Cy5

Figure AAE: Antibody Affinity Extraction (AAE).

Computerized Comparisons
The fluorescent CyDye images were analyzed using
Progenesis SameSpots software (version 4.5, 2011,
TotalLab, UK) and Progenesis PG240 software (version 2006, TotalLab, UK). The Cy3 and Cy5 images
are obtained from the same 2D gel and so are superimposable. Computerized analysis includes image
warping, spot outlining, background subtraction
(average on boundary), and quantification in conjunction with detailed manual checking. No differences were observed in the reverse labeling configuration (data not included) indicating the proteins
were not differentially labeled by Cy3 and Cy5.

sample. In experiments reported below, AAE columns were prepared using Cygnus Technologies 3G
goat anti-CHO HCP antibodies as employed in our
CHO HCP ELISA Kit (Cat# F550). A control column
was prepared using normal goat IgG. The following
conditions were tested:
1. CHO HCP passed over the 3G CHO HCP
column: Test condition
2. CHO HCP passed over the normal goat IgG
column: Non-specific binding control
3. E. coli HCP passed over the 3G CHO HCP
column: Non-specific binding control

Results

Silver Stain Analysis of Antibody
Affinity Extraction

Conventional
2D WB Analysis, Figures 1-3 CHO Harvest Material
Silver Stain versus CHO Harvest Material 2D WB
Detected by 3G Goat Anti-CHO HCP Antibody:

Duplicate gels were performed for the starting CHO
HCP sample and the AAE eluted HCP. The initial separation was performed by IEF in tube gels prior to
loading on the large format gel. The gels were run
at 25mAmp/gel for 5 hours. After washing, the gels
were silver stained and analyzed by computer analysis with manual checking.

Figure 1 is a silver stain (LF 845 #1) of the
commercial CHO harvest material. A total of
1293 unique spots were detected by either
silver stain or WB.

CoV2Research - May2020

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