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Multiplex fluorescent detection allows the detection and analysis
of multiple proteins on a single Western blot. This approach
facilitates quantification of proteins via the use of an internal
reference protein, removing the need to strip and reprobe a blot. The
FluorChem® Q provides a sensitive means to acquire high-quality
images of multicolor fluorescent Westerns. This report demonstrates
the sensitivity and linear range which can be achieved in using
the FluorChem® Q with ECL Plex™ labeled Westerns, and the
compatibility of the FluorChem® Q with multicolor Western blots.
Quantum Dot conjugated antibodies are powerful
tools that enable a multiplexed approach when
using standard Western blot assay techniques. The
ability to utilize optimized filter sets in conjunction
with high sensitivity fluorescent imagers significantly
enhances the capabilities of fluorescent detection
technology. Quantum Dot technology represents a
portfolio of powerful imaging reagents that are
compatible with both standard Western blot techniques
and the FluorChem® line of imagers available
from Cell Biosciences. In efforts to determine
the merit of using Quantum Dot technology to
quantify the detection of several proteins on a single
blot, we used routine procedures and methods for
detection of the phosphorylation levels of Inhibitor
of Kappa Beta Alpha (IκBα) in cell extracts using
both standard chemiluminescence and Quantum
Dot technology as detection reagents.
Gel imaging is a critical component in a wide variety of
today’s life science applications, and the need for clear,
precise, highly sensitive images is crucial. However, large
gel imaging systems often take up valuable lab space. Cell
Biosciences, a leading provider of high end chemiluminescence
and fluorescence imaging workstations, introduces the
red gel imaging system to provide quality imaging
capabilities to labs with limited space. The red system
is small enough to fit on a bench top, yet provides highly
sensitive and quantitative images for the most demanding
experiments.
Digital image acquisition using the FluorChem HD2
is compared to film for chemiluminescent Western
blot detection. The results show that digital imaging
produces higher quality images at equal exposure
times and achieves greater dynamic range and detects
lower levels of protein than film. With the additional
advantages of reduced cost and time expended
per image, image archiving, and quantitative image
analysis, digital imaging is the method of choice for
chemiluminescent Western blot detection.
The enzyme-drug, L-asparaginase, has been used since the 1970s to treat acute lymphoblastic leukemia. Asparagine synthetase (ASNS)
expression has been found to be correlated with L-asparaginase efficacy in leukemia cell lines, in leukemia primary tumor samples, and
more recently, in cancer cell lines from other tissues of origin. Silencing ASNS expression by RNAi has indicated the L-asparaginase/ASNS
relationship is causal and suggests that ASNS expression may be useful as a predictive clinical biomarker of L-asparaginase efficacy. ASNS
presents as a single peak in the NanoPro assay. Expected changes of expression are observed upon siRNA as well as L-asparaginase
treatment.
Lactate dehydrogenase (LDH) is a reversible enzyme that catalyzes the reduction of pyruvate to lactate or the oxidation of lactate to
pyruvate. In most human cells, LDH in its native form is a tetramer with 5 possible isoforms (LDH1-5), combinations of LDHA
(calculated pI: 8.4) and/or LDHB (calculated pI: 5.7). LDH overexpression and differential expression of LDH isoforms have been
implicated in the pathogenesis and progression of many cancers.
DNA analysis is a corner stone of molecular biology.
Electrophoresis of DNA in agarose gels is among
the most commonly used techniques for DNA
analysis. Internal standards can be incorporated into
DNA agarose gel experiments to maximize the
quantitative information acquired in the experiment.
DNA ladders are typically employed to determine
the molecular size (number of base pairs) of double
stranded DNA (dsDNA). The traditional method for
quantifying the mass of DNA in agarose gels has
been to extract the DNA from the gel in a series of
clean up steps (usually involving chloroform),
followed by a UV-VIS spectrophotometer reading.
AlphaQuant Molecular Ladders provide DNA
markers standardized for both molecular size and
quantity of DNA. This eliminates the need for
excision of DNA bands and extraction of DNA from
agarose gels. AlphaQuant Molecular Ladders
provide a convenient and fast method to determine
size and quantity of unknown DNA samples in
agarose gels.
Translation repressor protein 4E-BP inhibits cap-dependent translation by binding to the eIF4E translation initiation factor. Hyperphosphorylation
of 4E-BP disrupts this interaction and results in activation of cap-dependent translation. Both the PI3 kinase/Akt pathway and
FRAP/mTOR kinase regulate 4E-BP activity. 4E-BP1 has been implicated as a biomarker for several cancer types, while 4E-BP2 has been
shown to potentially play a role in energy homeostasis. We show 4E-BP1 activation in MCF10A cells in response to EGF using total and
anti-phospho 4E-BP1 antibodies that enable distinction between phospho and non-phospho peaks.
Translation repressor protein 4E-BP inhibits cap-dependent translation by binding to the eIF4E translation initiation factor. Hyperphosphorylation
of 4E-BP disrupts this interaction and results in activation of cap-dependent translation. Both the PI3 kinase/AKT pathway and
FRAP/mTOR kinase regulate 4E-BP activity. 4E-BP1 has been implicated as a biomarker for several cancer types, while 4E-BP2 has been
shown to potentially play a role in energy homeostasis. We show 4E-BP2 activation in MCF10A cells in response to EGF and 4E-BP2
inhibition in MCF7 cells with LY294002 (PI3 kinase inhibitor).
Crk-like protein (Crk-L) is an adapter protein and phosphotyrosine-containing substrate implicated in transformation by the bcr-abl
oncogene and in signaling by cytokines. It has been shown to activate the RAS and JUN kinase signaling pathways and transform
fibroblasts in a RAS-dependent fashion. Crk-L is a substrate of the BCR-ABL tyrosine kinase and plays a role in fibroblast transformation
by BCR-ABL. We show that Crk-L phosphorylation is reduced in response to Imatinib (commonly known as Gleevec®) treatment in
K562 cells.
Epitope tags are widely used for afinity purification as well as for highly sensitive detection of recombinant proteins. The c-Myc-tag
consists of a short peptide sequence (MEEQKLISEEDLLM). EGFP was expressed with a c-Myc-tag at either the C- or N-terminus of the
protein. As expected from the amino acid composition of this tag, a slight acidic shift in the pI of the tagged protein can be observed.
Epitope tags are widely used for afinity purification as well as for highly sensitive detection of recombinant proteins. The FLAG-tag
consists of a short peptide sequence (MADYKDDDDKM). EGFP was expressed with a FLAG-tag at either the C- or N-terminus of the
protein. As expected from the amino acid composition of this tag, a slight acidic shift in the pI of the tagged protein can be observed.
Epitope tags are widely used for afinity purification as well as for highly sensitive detection of recombinant proteins. The HA-tag consists
of a short peptide sequence (MAYPYDVPDYASM). Here, EGFP was expressed with a HA-tag at both the C- and N-terminal of the
protein. As expected from the amino acid composition of this tag, a slight acidic shift in the pI of the tagged protein can be observed.
Dual specificity mitogen-activated protein kinases (MEK) are members of the dual specificity protein kinase family, which act upstream
from the classical MAP kinases through phosphorylation and thus activation of ERK1 and 2 in response to a wide variety of extra- and
intracellular signals. While the functions of MEK1 and MEK2 are very similar, these kinases differ significantly in the way they are regulated.
For example, serum addition can specifically induce MEK1 activity in CHO cells. By contrast, MEK2 appears to be the functionally
predominant isoform in formyl-methionyl-leucyl-phenylalanine treated neutrophils. Here we show MEK1 activation in MCF10A cells
treated with EGF.