Opn final 10 19-10
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This document describes the development of monoclonal antibodies that target the DSVVYG peptide sequence of human osteopontin. Several antibody clones were selected and tested in ELISA and cell adhesion assays. Six antibody clones emerged as most promising based on their ability to detect recombinant and native osteopontin in ELISA and influence breast cancer cell adhesion. Further analysis is planned using these six clones, including western blotting of native osteopontin and additional cell adhesion assays using native osteopontin to better understand post-translational modifications.
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Lab Differential Expression Differential gene expression provides .pdf
Lab: Differential Expression Differential gene expression provides the ability for a cell or
organism to respond to a constantly changing external environment. The specific constellation of
proteins required for optimal function and growth varies with cellular age and environmental
context. Thus, protein production is carefully regulated by multiple mechanisms that modulate
both transcriptional and translational pathways. Control of transcription initiation by RNA
polymerase is a predominant mechanism for regulating expression of specific proteins,
presumably because it provides maximal conservation of energy for the cell. We can often
observe the consequence of differential transcription due to the presence or absence of particular
proteins or the growth in particular environments. Control can also occur at translation; the
mRNA is synthesized, but only in certain circumstances is it translated. Control can also occur at
the level of protein function; the protein is inactive, or activity is not observed due to the lack of
the substrate. In this lab we will observe differential expression of two different genes encoded
on plasmids. We will analyze transcriptional activity, translational activity, and protein function.
Plasmids are extra-chromosomal DNA. Bacteria often have plasmids and will replicate the
plasmid and pass it to daughter cells (vertical transmission) and to neighboring cells (horizontal).
Plasmids are a mechanism of gene diversity. In order to stably retain the plasmid, there needs to
be some type of metabolic reason for the bacteria to maintain the plasmid. In other words, the
plasmid confers an advantage. Plasmids contain: 1. Ori: the plasmid may present is low or high
copy number. 2. Lab generated plasmids typically also contain a selectable marker (antibiotic
resistance), 3. Additional gene for ease of visual screening 4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant plasmids. The transformed cells containing the plasmid with the gene of interest ca.
Preparing libraries directly from archived FFPE sections blood, saliva, and b...
Preparing libraries directly from archived FFPE sections blood, saliva, and b...Thermo Fisher Scientific
We have developed a research method to
prepare Ion AmpliSeq libraries directly from small
amounts of biological samples such as whole
blood, saliva, buccal swabs and even FFPE
sections.
• The direct library prep methods were automated
on the Ion Chef reducing user interaction to
loading reagents and samples on to the deck of
the Ion Chef.
• Sequencing metrics were comparable or
improved for the samples used directly without
purification as compared to purified DNA controls.
Additionally, as shown for buccal swab samples,
there was no detrimental effect on variant calling
results.
• Extremely small samples were successfully
processed with this method, which reduces the
total time from sample to sequence to 24 hours.ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE B.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE
BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides
the ability for a cell or organism to respond to a constantly changing external environment. The
specific constellation of proteins required for optimal function and growth varies with cellular
age and environmental context. Thus, protein production is carefully regulated by multiple
mechanisms that modulate both transcriptional and translational pathways. Control of
transcription initiation by RNA polymerase is a predominant mechanism for regulating
expression of specific proteins, presumably because it provides maximal conservation of energy
for the cell. We can often observe the consequence of differential transcription due to the
presence or absence of particular proteins or the growth in particular environments. Control can
also occur at translation; the mRNA is synthesized, but only in certain circumstances is it
translated. Control can also occur at the level of protein function; the protein is inactive, or
activity is not observed due to the lack of the substrate. In this lab we will observe differential
expression of two different genes encoded on plasmids. We will analyze transcriptional activity,
translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often
have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission)
and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to
stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to
maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori:
the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also
contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening
4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant.
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE BAC.pdf
ONLY THE LAST QUESTION IS THE POINT OF POST. THE OTHER PAGES ARE
BACKGROUND CONTEXT Lab: Differential Expression Differential gene expression provides
the ability for a cell or organism to respond to a constantly changing external environment. The
specific constellation of proteins required for optimal function and growth varies with cellular
age and environmental context. Thus, protein production is carefully regulated by multiple
mechanisms that modulate both transcriptional and translational pathways. Control of
transcription initiation by RNA polymerase is a predominant mechanism for regulating
expression of specific proteins, presumably because it provides maximal conservation of energy
for the cell. We can often observe the consequence of differential transcription due to the
presence or absence of particular proteins or the growth in particular environments. Control can
also occur at translation; the mRNA is synthesized, but only in certain circumstances is it
translated. Control can also occur at the level of protein function; the protein is inactive, or
activity is not observed due to the lack of the substrate. In this lab we will observe differential
expression of two different genes encoded on plasmids. We will analyze transcriptional activity,
translational activity, and protein function. Plasmids are extra-chromosomal DNA. Bacteria often
have plasmids and will replicate the plasmid and pass it to daughter cells (vertical transmission)
and to neighboring cells (horizontal). Plasmids are a mechanism of gene diversity. In order to
stably retain the plasmid, there needs to be some type of metabolic reason for the bacteria to
maintain the plasmid. In other words, the plasmid confers an advantage. Plasmids contain: 1. Ori:
the plasmid may present is low or high copy number. 2. Lab generated plasmids typically also
contain a selectable marker (antibiotic resistance), 3. Additional gene for ease of visual screening
4. Multiple cloning site
pUC19 is one of a series of plasmid cloning vectors created by Joachim Messing and co-workers.
The designation "pUC" is derived from the classical "p" prefix (denoting "plasmid") and the
abbreviation for the University of California, where early work on the plasmid series had been
conducted. It is a circular double stranded DNA and has 2686 base pairs. pUC19 is one of the
most widely used vector molecules as the recombinants, or the cells into which foreign DNA has
been introduced, can be easily distinguished from the non-recombinants based on color
differences of colonies on growth media. pUC18 is similar to pUC19, but the MCS region is
reversed. - pUC 19 has an origin of replication and is maintained at a high copy number. -
pUC19 encodes for an ampicillin resistance gene (amopR), via a -lactamase enzyme that
functions by degrading ampicillin and reducing its toxicity to the host. - It has an N-terminal
fragment of -galactosidase (lacZ) gene of E. coli which allows for visual screening of
recombinant.
Similar to Opn final 10 19-10 (20)
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Preparing libraries directly from archived FFPE sections blood, saliva, and b...
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Opn final 10 19-10
- 1. Monoclonal Antibodies Specific for DSVVYG Peptide of Human Osteopontin
- 2. Functional Domain Structure of Osteopontin Immunogen to target cryptic site of OPN: DSVVYG
- 3. Assays: Indirect ELISA Peptide Recombinant N-term OPN Recombinant Full Length OPN Native OPN (purified from human breast milk), evaluated on SDS-PAGE
- 4. Clones Selected for Further Study Summary of Indirect ELISAof Selected Clones 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 03H05 09C11 10E02 11H05 13F04 14C08 15C02 16A05 16A11 Clone designation A(450) DSSVYG-BSA recN-terminal OPN recflOPN Native OPN
- 5. Assays: Sandwich ELISA Nine selected peptide clones as tracer with current 5 MABS as capture, rec-flOPN and native OPN as antigen Nine selected peptide clones as capture with current 5 MABS as tracer, rec-flOPN and native OPN as antigen
- 6. Matched Pair ELISA, Peptide clones Tracer Match Pair Assay Panel with OPN sandwich 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 MAB193P MAB194P MAB195P MAB196P MAB197P nocoat MAB193P MAB194P MAB195P MAB196P MAB197P nocoat Capture MAb's ODvalue 03H06 09C11 10E02 11H05 13F04 14C08 15C02 16A05 16A11 MAB197 no biotin Native OPN flOPN
- 7. Matched Pair ELISA, Peptide clones Capture Match Pair Assay Panel w/ OPN Sandwich 9-1-10 MH 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 03H06 09C11 10E02 11H05 13F04 14C08 15C02 16A05 16A11 MAB193 nocapture 03H06 09C11 10E02 11H05 13F04 14C08 15C02 16A05 16A11 MAB193 nocapture Capture Ab's Absorbance450nm MAB193P MAB194P MAB195P MAB196P MAB197P no biotin Native OPN flOPN
- 8. Cell Adhesion Assays Breast Cancer Cell Line: MDA-MB-231 Percent Cellular Adhesion 0 20 40 60 80 100 120 09B05 16A09 16E12 irrelevant Ab No Mabs Antibody Percent N-terminal FL-OPN
- 9. Conclusions MBS has developed a panel of antibodies specific to the unique DSVVYG sequence of OPN Six clones emerged based on the data generated from ELISA and Cell Adhesion as being of most interest. Data demonstrates that using native OPN in the assays is optimal, as recombinantly expressed OPN in E. coli will not provide the post translational modifications of native OPN Samples of the six clones are available upon request
- 10. Next Actions Study of Peptide specific OPN clones by Western Blot of Native OPN Study of Peptide Specific OPN clones on Cell Adhesion Assays using Native OPN References: Alicia Plumer, Hongyi Duan, Sripriya Subramaniam, F Lee Lucas, Susan Miesfeldt, Ah- Kau Ng, Lucy Liaw. Development of fragment specific osteopontin antibodies and ELISA for quantification in human metastatic breast cancer BMC Cancer 2008, 8:38 (31 January 2008)
Editor's Notes
- Additional studies currently underway include a western blot analysis using native purified OPN against all six of the selected clones as well as cell adhesion assays using the native OPN. If the DSVVYG peptide is linear, the western blot data will shed light on the integrity of the purified native OPN by immunostaining full length and /or OPN fragments containing the DSVVYG, indicated by molecular weight of bands. Additionally, the ability of these clones to inhibit cell adhesion by binding to native OPN will be of significant value to cancer researchers and is the motivation to continue these studies. Results of the extended studies will be made available at a later date. Please contact Sales to access samples of this unique panel of Osteopontin Monoclonal antibodies.