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DETECTprotein binding pair-
Analytical detection by high throughput screen
Conditions can be varied simply, cheaply
CONFIRMmolecular interaction at the structural level-
Biochemical characterization by IP / MS
ESTABLISH biological significance-
Confirmed by the identification of intracellular interactions
that are crosslinked in vivo,
then characterized as above
DETECTprotein binding pair-
Analytical detection by high throughput screen
Conditions can be varied simply, cheaply
Part I
Suggestion– there is a modification to a well-established
Q-ELISA format, that would allow for a screen for interacting
proteins. First, recall a Quantitative ELISA format -
Trap protein of interest with a monoclonal AB, allow-
ingthe protein to be uniformly oriented and mostly free
Bathe the mAB with your physiological fluid or
culture isolate of choice
Detect protein of interest with a polyclonal AB, allowing the
protein to be bound with multiple AB molecules to optimize
the signal from biotin
Detect the protein of interest with sensitive laser and
~irreversible formation of signal
Suggestion–
using this same Q-ELISA based
format, with the monoclonal
capture antibody, and a 2nd
detection antibody, one can
change the detection antibody to
an AB (Layer 3) that recognizes
the interaction partner to identify
the macromolecular complex,
and characterize the conditions
under which the complex is
present or absent.
Uses standard clinical assay Q-ELISA based instrumentation,
developed for high throughput, minimal sample volume, has
been verified by numerous assays for several antigens, by
commercial kit or customized design
Kits already in use
Apoptosis
Signal transduction, AKT pathway
Acute phase response panel
Inflammation panel
Death receptor pathway
Growth factors
Lipoprotein panel, cardiac evaluation
•mAB to lipoproteins for the capture step
•pAB –biotin for the detection step
•Standardized control apolipoprotein (conc. known)
•Construct beads with mAB attached
•Run the assay using the standardized control
apolipoprotein to confirm that the reagents are working as
expected.
•Run the assay using unknown sample, and control sample
for the standard curve.
Detection of protein or protein complex is determined by
the selection of trapping and detecting antibody pairs
*Quantitative difference likely due to
(1) Change in C1q level due to loss associated with blood clotting
and clot removal from the serum, or
(2) an increase in accessibility of the C1q epitopes when the protein
is free (plasma) v. in complex, (serum), since C1s, C1r coat the 6
arms of the C1q molecule.
Quantization of C1s is similar for free v. complexed protein
C1s is free in plasma, in the C1 complex in serum
Single component detection
***Since the C1s concentration does not
change appreciably between serum and plasma, it is likely that
(2) is the reason for the change in C1q concentration.***
Examples for the components of C1 and the C1 complex of 3 proteins
Detection
Capture /
trapping
Information
Yield
Q S R
Q S R
Q QQ
C1q C1 C1
Examples for the components of C1 and the C1 complex of 3 proteins
Detection
Capture /trapping
Information Yield
Q S R
Q S R
S SS
C1 C1s C1r2C1s2
Part II
CONFIRMmolecular interaction at the structural level-
Biochemical characterization by IP / MS
Biochemical proteomics
Further molecular analysis at the biochemical level-
1. magnetic beads to capture the immune complex,
2. followed by further screening, including MS
AB-AG
Part III
ESTABLISH biological significance-
Confirmed by the identification of intracellular interactions that
are crosslinked in vivo, then characterized as above, Part I, II
Intracellular ID of complex, using biosynthetically labeled proteins with highly
reactive and nonspecific AA chemistry, either diazirine- M, or L, or acetylene-M.
Protein – protein interactions that change to initiate, or, as a result of, signal transduction
-using physiological conditions, screen for signal - specific interactions-
such as ligand binding, metal binding, oxidative stress, Calcium concentration,
low Oxygen tension, other pathway signals, that will change on signal transduction,
or with the onset of pathophysiology.
Document the correlation between signal and the protein – protein interaction in question.
Determine the physical factors that allow optimal complex formation, rapidly,
with a minimal amount of material, and under a range of conditions.
1. Screen recombinant proteins v. endogenous, native proteins.
2. Determine if an antibody that is generated to a peptide, can then
recognize the folded domain or protein.
3. Screen proteins that have been altered by engineering,
mutation, or chemical changes
4. Optimize conditions to study the interaction of interest.
It is possible to use a tag antibody, such as anti-His, to produce
a general screen for protein – protein interactions where one
partner is not known. Furthermore, it is possible to use an
antibody that targets a specific chemical modification, such as
biotin, thereby allowing for screening of a mixture of unknown
proteins for either the capture or detection sample.
Part I1. Assay format (96 well plate, complex formation on beads, streptavidin-biotin detection, laser
detection, single bead analysis) is well established. Adequate technical support is available.
2. Assay design has been validated on a complex that is well understood, from both a physiologic
standpoint, and a biophysical standpoint.
3. Assay format is suitable for complex mixtures, in a physiologic background.
4. Assay design is viable for proteins that are minor components of the protein milieu.
Part IIThis aspect of the program is in place to allow biochemical and biophysical confirmation of
complex formation. It capitalizes on the optimization screening that is carried out in Part I, by
using a similar format (immunocapture on beads), but in the larger scales required for
biochemical and biophysical analysis, in comparison to laser based fluorescence detection.
Part III1. Familiarity with the nuances of crosslink formation in proteins is in place from previous work.
2. Cell biology is in place from previous work.
3. Assays from Part I can be used to screen for crosslink formation, again requiring small
amounts of material for reliable screening, to compensate for possible low yields @ the
crosslink and harvesting steps.
4. Methods do not require radioactivity.