Cytiva eBook - 18
Confidence in host cell protein (HCP) coverage assays with differential gel approaches
2D SDS-PAGE/Western blot
AFFINITY + DIGE
DIBE™ technology
Total proteins labeled
with CyDye™ Cy3
2D SDS-PAGE
Cy3
Labeled
proteins
Cy3
Captured protein
labeled with Cy5
Cy5
Anti-HCP Ab + Cy5
detection reagent
fluorescent
detection
ECL™
reaction
detection
Western blot image
(colorimetric and
chemiluminescence)
Cy3
Cy5
Image
(detected HCPs)
CONFIDENCE
Confidence
COVERAGE
Coverage
50%
75%
| GENengnews.com
Cy3
+
Cy5
100%
LOW
MEDIUM
HIGH
50%
75%
Overlay image
CONFIDENCE
Confidence
Cy3
Coverage
+
Cy5
Confidence
86%
100%
Using two gels doubles the time
and effort needed
Using only one gel reduces
time and effort needed
2D spot comparison is
challenging and not standardized
No need for affinity column
Coverage values can vary within
and between labs
Immunodetection can be
more sensitive than affinity
and DIGE approach
Immunodetection in
denatured conditions
High-resolution = accurate
identification of all signals
cytiva.com/HCP-analysis
Cy5 Image
(detected HCPs)
Analysis in
Melanie
software
86%
Provides good separation
of HCPs
18
Overlay image
COVERAGE
Coverage
57-86%
+
Cy3 Image
(total protein)
Analysis in
Melanie™
software
Overlay image
Cy5
+
Cy3 Image
(total protein)
Analysis in
imaging
software
+
Labeled proteins
Detection
Detection
1˚ ANTI-HCP
+
2˚ HRP
conjugated
Detection
Western
blot
Unbound
protein
discarded
Transfer to
membrane
Transfer to membrane
Total protein
stain (IN-GEL)
Anti-HCP Ab column
Total proteins
labeled with Cy3
LOW
MEDIUM
Immunodetection in
denatured conditions
HIGH
50%
75%
100%
LOW
MEDIUM
HIGH
HCP capture in
native conditions
May under represent tightly
bound HCPs
Avoids challenges of
immunoblot transfer
Requires careful preparation of
anti-HCP resin which may be
difficult to reproduce
May not be as sensitive as a
Western blot
Cytiva and the Drop logo are trademarks of Global Life Sciences IP Holdco LLC or an affiliate. Cy, DIBE, and ECL are trademarks of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva.
Melanie is a trademark of SIB Swiss Institute of Bioinformatics. CY8146-08Jun20-IG
https://www.cytivalifesciences.com/solutions/protein-research/hcp-analysis?extcmp=CY20363-GL-GCR-PD-HCPprelaunchEbookGEN-exteml-exteml-ebook
Cytiva eBook
Table of Contents for the Digital Edition of Cytiva eBook
Contents
Cytiva eBook - 1
Cytiva eBook - 2
Cytiva eBook - 3
Cytiva eBook - Contents
Cytiva eBook - 5
Cytiva eBook - 6
Cytiva eBook - 7
Cytiva eBook - 8
Cytiva eBook - 9
Cytiva eBook - 10
Cytiva eBook - 11
Cytiva eBook - 12
Cytiva eBook - 13
Cytiva eBook - 14
Cytiva eBook - 15
Cytiva eBook - 16
Cytiva eBook - 17
Cytiva eBook - 18
https://www.nxtbookmedia.com