EXPERTise IN CELL & MOLECULAR BIOLOGY, BIOCHEMISTRY, BioPHYSICAL CHEMISTRY, GENETICS, MICROBIOLOGY

MarinBio’s expert team blends microbiology, genetics, biochemistry, biophysical chemistry and cell biology expertise with molecular biology and product development experience to achieve rapid results in a wide range of applications. Whether it is identifying a specific gene, manipulating or cloning a gene or producing and characterizing a protein drug product from a cloned gene or identifying/characterizing a drug metabolite, MarinBio makes it happen successfully for contracting clients.

Western Blot (left panel): Proteins were electrophoresed, transferred to PVDF membrane and detected with a specific primary antibody and a secondary antibody- HRP with the signal developed with BCIP/NBT chromogenic substrate.

Southern Blot (right panel): DNA from different maize plants was digested with Kpn1 or EcoRV, electrophoresed and probed with mcry3a probe labeled with Digoxigenin (replaces 32P probes) resolving clear band.

Western Blot

Digoxigenin (DIG) Non-radioactive
Detection of Southern Blot

CELL & molecular biology, MICROBIOLOGY

MarinBio’s professional scientists are expert at analysis and manipulation of cells, genes, protein drugs or other biologics and small molecule or large molecule pharma or natural product drugs including, as follows:

  • Drug or Agent Minimum Inhibitory Concentration (MIC)
  • Phage display to identify high affinity and selective protein sequences
  • Plasmids and recombinant viral vectors
  • Gene expression, QRT-PCR, QPCR, Real Time-PCR, Southern & Northern blots
  • Reporter assays
  • siRNA gene silencing and other gene regulation (see gene expression below)
  • Transfection & lipofection – electroporation / viral-based, lipid-based, cationic, transient, stable cell lines; see also transfection and exosome pages
  • Vector construction

QUICK READS

From the beginning and ongoing, your assay is a critical component of the life cycle of your current or eventual product. That is precisely why you want experience and expertise at the earliest possible stage of your development.

MarinBio has special expertise in the Polymerase Chain Reaction (PCR) as extremely versatile techniques for copying DNA or RNA molecules. PCR enables a specific nucleic acid sequence to be copied or modified, where a single DNA or RNA molecule is amplified to a billion cloned identical molecules in less than two hours by PCR via thermal cycling. We also have experience with isothermal PCR gene amplification which is enzymatically driven at room temperature. DNA PCR determines whether a particular DNA fragment is found in a cDNA library. At MarinBio, DNA PCR is also successfully employed, for example, to introduce specific restriction enzyme sites to the ends of DNA molecules or to mutate particular DNA bases in site-directed mutagenesis. Reverse Transcriptase-PCR (RT-PCR) amplifies RNA. Quantitative PCR (QPCR) of DNA and Quantitative RT-PCR of RNA enables quantitative measurements for determining precise amounts of DNA or RNA molecules, respectively.

In agarose gel electrophoresis, DNA and RNA are separated on the basis of size by migrating the DNA through an electrically charged agarose gel. Proteins are separated on the basis of size by using a SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis, or on the basis of size and their electric charges via 2D (2 Dimensional) gel electrophoresis. The DNA, RNA and protein macromolecules are detected by a specific probe (e.g. a sequence specific nucleic acid probe for DNA or RNA sequences or a specific antibody probe for a protein encoded antigen) after blotting the molecules onto an appropriate membrane support.

Southern blot probes for the presence of a specific DNA sequence within a DNA sample. DNA samples, before or after DNA restriction endonuclease enzyme digestion, are separated by gel electrophoresis and then transferred to a membrane support by blotting via capillary action or other transfer techniques. The membrane is then exposed to an appropriate labeled probe with a complementary base sequence to the sequence of interest. At MarinBio, Southern blotting is still in use for certain client applications, for example in measuring transgene copy numbers in transgenic mice or in the engineering of gene knockouts in embryonic stem or other cell lines.

Northern blot is essentially a combination of denaturing RNA gel electrophoresis and a blot to a support membrane. In this Northern blot process, RNA is separated based on size and is then transferred to a membrane that is then probed with a labeled complementary sequence. The results are visualized, depending on the label used; however, most result in various RNA bands representing the sizes of the RNA detected in the sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed. This procedure is commonly used by MarinBio and others to monitor when and how much gene expression is occurring by measuring how much of that RNA is present in different samples. Northern blotting is a common technique for determining at what time, and under what conditions, certain genes are expressed in living cells and the effects of drugs on RNA expression.

Eastern blots detect post-translational modification of specific proteins. Proteins blotted on to PVDF or Nitrocellulose membranes are probed for specific modifications using appropriate specific substrates.

In Western blots, proteins are first separated by size, typically in a thin gel sandwiched between two glass plates in SDS-PAGE (described above). The proteins in the gel are then transferred to a PolyVinyliDene Fluoride (PVDF) membrane, nitrocellulose, nylon, or other support membranes. The membrane is probed with antibodies that specifically bind to the protein of interest and are visualized, for example, by fluorescence, chemiluminescence or autoradiography. Enzyme detection is enabled when the antibodies are labeled with an enzyme reporter and the respective chemiluminescent substrate. Western blotting techniques allow not only detections but also quantitative analyses. Analogous protein Slot blot methods to Western blotting are used by MarinBio and others to directly stain specific proteins in live cells or tissue sections.

biochemistry & BIOPHYSICAL CHEMISTRY: Recombinant PROTEIN PRODUCTION, purification, CHARACTERIZATION & Stability

When MarinBio clients require designer protein product production, purification and characterization from transfection producing transient or stable cell lines, our staff PhD scientists have many years of experience and successfully perform these client projects. Our niche is to take cross scientific field projects in molecular biology and biochemistry, biophysical chemistry, genetics, microbiology and cell biology in bringing our breadth of expertise to successfully complete client projects, for example, as follows:

  • Recombinant protein production in cultured cells –bacterial/plasmid vectored cells, –insect cells/Baculovirus vectored, –yeast cells, –mammalian cells
  • Inducible cell or viral vectored gene promotors and specific cell signaling factors for high level protein expression
  • Protein size, charge, conjugation, stability; Western blots for proteins and Eastern blots for post-translational modifications
  • Chromatography, electrophoresis, charge variant analyses
  • Characterization: Protein size, charge, pI (Isoelectric point, pH where net charge on protein is zero), stability, Eastern blots for post-translational modifications
  • Western blots, ELISAs, proteomics, drug binding protein assays
  • Enzyme assays, protein assays, reactive oxygen species assays
  • Ligand binding, affinity, inhibition, metabolic and radioisotope assays
  • Native protein purifications from plasma, cells, tissue extracts

 

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