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
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