The Most Interesting Scientist in the World


The Most Interesting Scientist in the World

 

Camilo Moncada, Ph.D. is a senior scientist and Director of QC at Rockland Immunochemicals, a leading company in antibody development. Dr. Moncada began his research studies over 10 years ago and has joint publications in different subjects including immunology, parasitology, cancer and lung disease. At Rockland, he applies his expertise on molecular and cellular biology, biochemistry and proteomics for antibody development and validation. You can find his tips and recommendations on a variety of applications below.

 


Tips for Optimal SDS-PAGE Separation

 

Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) is used to separate mixtures of proteins of variable complexity and allows you to determine sample protein composition, purity, whether you have a target of interest in your sample, and even some of its structural characteristics. Gel electrophoresis is a basic tool in itself, but also is the stepping stone to other important techniques in the lab going hand in hand with western blotting (WB). You can't have a good western blot without first having a good SDS gel. In this particular tips segment, we are discussing protein gel electrophoresis using precast gels and the parameters involved. Below are some pointers for optimizing your SDS-PAGE results. 

 


Tips for Multiplex Fluorescent Western Blotting

 

Fluorescence based multiplex Western blot is suited for simultaneous detection and quantification of specific proteins in a biological sample. Using a combination of two or three antibodies, fluorescent detection enables simultaneous quantitative analysis of multiple proteins within the same sample on the same blot.  

 


Fit-for-Purpose Antibodies

 

Antibodies have become essential tools for research, diagnostic and therapeutic purposes because of their high specificity, high binding affinity, long half-lives and low toxicity. Antibodies comprise those secreted by a single clone of B lymphocytes, termed monoclonal antibodies, and those produced by a mixture of various B lymphocyte clones, termed polyclonal antibodies. Antibodies are invaluable reagents for antigen detection and purification, e.g. immunoblotting, immunoprecipitation, immunohistochemistry, ELISA, and immunoaffinity chromatography. Successful production of antibodies depends upon careful planning and implementation of critical steps that may influence the outcome of the effective antibody responses.

 


Tips for Immunoprecipitation

 

Immunoprecipitation (IP) is a well-established technique used to isolate a specific protein or group of interacting proteins from a complex mixture of many different proteins using an antibody immobilized on a solid support. These solutions are often in the form of a crude lysate of cells, an animal tissue or a plant. IP is an important step in many proteomic studies designed to explore the presence, relative abundance, protein function, protein-protein interactions, post-translational modifications and expression profiling of proteins. Purified proteins obtained by immunoprecipitation can be analyzed by variety of techniques, such as ELISA and Western blotting

 


Tips for Optimizing Protein Expression and Purification

 

Recombinant proteins are used throughout biological and biomedical science. The development of simple, commercially available systems has made the production of recombinant proteins more widespread. Most significantly, it has dramatically expanded the number of proteins that can be investigated both biochemically and structurally. Since every protein is different, the purification protocols and strategies must be worked out for each individual protein and with an eye to its intended use. We describe the various factors that have a large effect on soluble protein expression and describe how to change them in order to express folded, active proteins.


Antibody Purification Using Immobilized Protein A and Protein G

 

The basis for purification of IgG, IgG fragments and IgG subclasses is the high affinity of protein A and protein G for the Fc region of polyclonal and monoclonal IgG-type antibodies. Protein A and protein G are bacterial proteins, which, when coupled to chromatography matrix such as Agarose or Sepharose, generate exceptionally useful, easy to use media (resin) for many routine applications. Protein A/G is a recombinant of Protein A and Protein G that has the additive binding properties of both proteins. Protein A, protein G and protein A/G can be used for purification of monoclonal IgG-type antibodies, purification of polyclonal IgG subclasses, and the adsorption and purification of immune complexes involving IgG. IgG subclasses can be isolated from cell culture supernatants and serum and from ascites fluid.

 


Diluting Antibodies: Technical Tips

 

Western blotting and immunohistochemistry (IHC), antibody titer and dilutions are important for their effect on signal and staining quality. Correct dilutions of antibodies will contribute to the quality of signal/staining if prepared precisely and consistently.

 


 Immunohistochemistry: Technical Tips

 

The acceptability of scientific results depends on the accuracy and sensitivity of the appropriate methods used, as well as controlled specificity of reagents and processes. This is particularly valid for immunohistochemistry, where sensitivity and specificity of the antibodies, as well as methodological procedures are critical to avoid false-positive and false-negative results.  

 


Flow Cytometry: Technical Tips

 

Flow cytometry is a technology that allows rapid and quantitative multi-parameter assays on single living (or dead) cells by measuring visible and fluorescent light emission signals.  

 


Western Blotting: Technical Tips

 

Western blot or immunoblotting is a rapid and sensitive technique that uses antibodies for the specific detection of proteins separated by polyacrylamide gel electrophoresis (PAGE) and immobilized onto a nitrocellulose, nylon or PVDF membrane.

 


ChIP: Technical Tips

 

Chromatin immunoprecipitation (ChIP) is a powerful technique to determine protein interactions at particular regions of DNA in order to map the relative position of chromatin and DNA binding proteins such as histone modifications.

 


Immunofluorescence Microscopy: Technical Tips

Immunofluorescence microscopy (also known as IF microscopy) is a useful technique for detection and localization of cellular proteins and other antigens via fluorescent-labeled antibodies. 

 


ELISA: Technical Tips

 

ELISA represents one of the most widely used antibody applications from basic research to diagnostics. This assay is the preferred method to determine the titer of an antibody but can also be successfully used to quantitative antigen or analytes in a sample.  

 


Biotin, Avidin, Streptavidin: Technical Tips

 

The (strept)avidin-biotin system is a protein-ligand interaction present in nature that has been successfully used in a number of applications including detection of proteins, nucleic acids and lipids as well as protein purification. 

 


Tips for Selecting the Best Secondary Antibody

 

Antibody-based assays or immunoassays represent a widely used, valuable tool in areas of basic research, bioprocessing, diagnostics and clinical applications. Although successful detection of the target protein relies on multiple parameters, it is well recognized that the use of high quality antibodies critically affects assay performance.

 


Tips for Culturing Human Melanoma Cell Lines

 

Rockland Immunochemicals has partnered with The Wistar Institute to produce, validate and distribute a diverse panel of Human Melanoma Cell Lines. Trish Brafford, a Wistar research assistant, has worked exclusively with the cell lines and is here to provide tips for culturing the cells to their full potential.

 


Mastering Post-Translational Modifications

 

Post-translational modifications (PTMs) play a key role in dynamic cellular processes, regulating gene expression, protein activity, localization, and degradation, as well as protein interaction. In this poster, we have teamed up with The Scientist to show you the most important tips for choosing the best high-affinity, high-specificity antibody for your PTM detection needs.

 


Tips for TrueBlot® Immunoprecipitation/ Western Blotting

 

We provide guidelines for detection of immunoprecipitated samples by Western blotting utilizing TrueBlot® immunoblotting reagents. These readily available reagents provide flexibility to accommodate proteins with overlapping molecular weights to the heavy and light chain fragments in the IP experiments. These options offer cleaner WB membranes and therefore target signals that are simple to interpret.

 


Tips for Selecting Conjugated Antibodies

 

Antibodies are used to detect and quantify antigens using an appropriate detection technique such as flow cytometryELISA,Western blottingimmunofluorescence and immunohistochemistryLabeling strategies result in the covalent attachment of molecular labels to the target protein in order to facilitate the detection of a labeled protein and its binding partners. While multiple types of labels are available, their diverse uses are preferable for specific applications. Therefore, the type of label and the labeling strategy used must be considered carefully and tailored for each application.

 


Tips for Collagen

 

Collagens represent a large family of proteins which are the essential components of the extracellular matrix, the chief structural component of skin and the most abundant protein in the human body representing approximately 30% of the total dry weight. Collagen is an attractive substance for various medical applications, such as implants, transplants, organ replacement, tissue equivalents, vitreous replacement, plastic and cosmetic surgery, surgical suture and dressings for wounds and burns among others.



ELISA Kit Tips


Rockland offers a wide variety of ELISA kits in sandwich assay formats. This extensive portfolio includes over 800 complete, ready-to-use ELISA kits for detection of specific growth factors, cytokines, chemokines, hormones, glycoproteins, and neurotrophic factors that are common targets of research interest. Each enzyme linked immunosorbent assay is rigorously tested to ensure high precision, accuracy, sensitivity, and specificity. Each ELISA kit is optimized for accurate quantitation of targets in various sample types, including serum, plasma, saliva, cell lysates, and cell culture supernatants.

 

 
 

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