Antibody News

Antibody Detection On Western Blot Membranes

Monday, December 28, 2009 - 10:00

Western blotting combines gel electrophoresis with use of a membrane to separate and identify target proteins using antibodies. Proteins are separated into bands using electrophoresis, and are then transferred to a membrane using filter-paper capillary action or an electroblotting technique. The effectiveness of the transfer can be checked by means of a stain – typically Ponceau S.

The membrane is then blocked from further protein interaction with diluted bovine serum albumin or non-fat milk solution. This ensures the antibodies bind only to the proteins, and not to the membrane substrate. False positives and fuzzy results are thus minimized, giving coherent and repeatable results.

The next stage is to incubate the membrane with a diluted primary antibody solution - typically 0.5 to 5.0 mcg/mL. The membrane is rinsed to remove any unbound protein, and exposed to a secondary...

An Overview Of The Western Blot Immunoassay

Friday, December 25, 2009 - 10:00

Western blot is a widely used immunoassay technique, used to identify proteins. Many people enter Western blot research without having a clear idea of how the technique relates to antibody usage, so we at Novus Biologicals thought it would be interesting to give a general review of this subject.

Western blotting, also called protein immunoblotting, uses gel electrophoresis to separate proteins of tissue extracts and homogenates. Native and denatured proteins are identified by their isoelectric point, electric charge, molecular weight or molecular structure (often, a combination of these).

Tissue for Western Blot analysis is blended or homogenized to break down cells and release the proteins. Bacterial and viral proteins are also identified using Western blot, and samples are not restricted to cellular tissues...

Embryonic Stem Cells No Longer The Only Option In Antibody Research

Wednesday, December 23, 2009 - 10:00

Human stem cell research has proved a controversial topic, but the latest developments could alter the debate significantly.

Among the products in our antibody database at Novus Biologicals, we have a range of stem cell marker immunoglobulins. PODXL antibodies, also known as TRA-1-60 and TRA-1-81, which fall under this category of stem cells, are widely used in cancer research. PODXL is known to be involved in the development of several aggressive cancers, among them pancreatic, breast and prostate cancer. It was first identified on the cell surfaces of epithelial cell lines.

PODXL antibodies, such as our TRA-1-81 and TRA-1-60 antibodies, are specific to human cell lines, with no immunoreactivity to murine cells being seen. Typically, this means using human embryonic stem...

Using Recombinant Proteins In Antibody Research

Monday, December 21, 2009 - 10:00

Recombinant proteins are those derived from recombinant DNA. Recombinant DNA is an artificial form of DNA, made by combining protein sequences which would not occur in their natural state.

Recently, we at Novus Biologicals have been developing recombinant bacterial proteins to reproduce existing products on our antibody database. Recombinant proteins are created from complementary cDNA libraries, which contain only DNA sequences that code for specific proteins. When this cDNA is expressed as a recombinant protein, it can be used in its purified form to perform studies into, for example, enzyme kinetics and NMR. The protein can also be used to create antibodies for further research – something which has been keeping our lab staff busy in recent times!

To take this further, if DNA sequences for certain genes are unavailable or not known, antibodies could be used for screening instead....

Fluorescent Dyes And Conjugated Antibodies

Friday, December 18, 2009 - 10:00

Antibody conjugation involves tagging on a protein, compound or dye, in order to track its interaction with specific antigens. It is a routine procedure in immunoassays. We at Novus Biologicals have a vast number of products in our conjugated antibody catalog, to which we are constantly adding.

One of the most exciting developments of recent years has been tagging with fluorescent dyes, such as Alexa and DyLight fluor. For example, Beta-actin antibody conjugated to DyLight 549 is used in immunofluorescent assays, using a synthetic peptide conjugated to KLH (Keyhole Limpet Haemocyanin) as the immunogen.

There are several DyLight products, categorized by a number which relates to the spectrum in which they absorb and emit light. DL 549 absorbs light at 562 nm and emits it at 576 nm. Dylight 549 labeled antibodies fluoresce with a yellow...

Explaining Conjugated Antibodies

Wednesday, December 16, 2009 - 10:00

A conjugated antibody (also known as a tagged, loaded or labeled antibody) is one which has been attached to a substrate such as an enzyme, toxin or inorganic compound. Modern immunoassay techniques make extensive use of proteins conjugated with fluorescent dye.

Contemporary antibody sources sometimes supply a wide range of both polyclonal and monoclonal conjugated antibodies. For the benefits of those who are new to this area, we thought it would be a good idea to provide a synopsis on what these proteins actually are, how they work and how they are used.

Conjugated antisera are used in many research applications, from Western blotting to in vivo cellular analysis. Researchers are looking for two things – a strong signal and specificity to particular antigens. We at Novus Biologicals pride ourselves on having an extensive catalog of highly specific products, including antibodies tagged...

Bcl-2 Antibodies And Apoptosis Research

Monday, December 14, 2009 - 10:00

Bcl-2 antibodies are used as markers for the Bcl-2 gene. Bcl-2 suppresses apoptosis (controlled cell death) by inhibiting caspase activity and controlling cell membrane permeability. It does this by forming mitochondrial pores on the outer membrane of the mitochondria. However, its exact mechanism of action is still not completely understood.

There are more than 25 Bcl-2 proteins, which are membrane bound and regulate apoptosis both positively and negatively. We at Novus Biologicals have a broad antibody database specific to the Bcl-2 gene, as well as anti-Bax antibodies. These two proteins are closely related, as Bax is a pro-apoptotic protein of the Bcl-2 family, forming channels in lipid membranes.

In planar lipid bilayer experiments, Bax formed ion-conducting channels which were both voltage and pH dependent. Further studies used Bax to trigger...

GTPase Regulators And Their Antibodies

Friday, December 11, 2009 - 10:00

Researchers routinely use GTP binding antibodies for the research of human disease. Thus it is worthwhile to provide a recap of the GTP binding process.

GTPases, a type of heterotrimeric G proteins, are essential to all metabolic functions. They act as molecular switches on the cell membrane, activating in response to various chemical signals. For example hormones, cytokines, physical stresses and cell-to-cell communications all activate downstream pathways. The interactions are complex, each requiring a different GTP protein. These function as molecular switches to activate or deactivate specific cellular responses.

G-proteins work by hydrolyzing GTP (guanosine triphosphate). They switch between active and inactive states, depending on whether they are GTP (guanosine triphosphate) or GDP (guanosine diphosphate) bound. In their active (GTP) state, they interact with effectors to initiate a downstream signal. In their GDP state...

Use Of FANC Antibodies To Study The Fanconi Anaemia Pathway In F And D1 cells

Wednesday, December 9, 2009 - 10:00

Fanconi anaemia (FA) is an autosomal-recessive disorder, characterized by skeletal abnormalities, progressive bone marrow failure and cellular hypersensitivity to DNA damaging agents such as Mitomycin C. FA also increases the likelihood of cancerous tumors and childhood leukemia.

Fanconi anaemia (FA) is a known complication of cancer therapy, and the use of FANC antibodies form a routine part of cancer research. Antibody suppliers, such as Novus Biologicals, supply anti-FANC antibodies which interact with FANC proteins in Western blot analysis and similar assays.

The FANCD2 protein is of particular importance, and is activated via the interaction of several other FANC proteins in a nuclear complex. This process (known as monoubiquitination) results in the formation of FANCD2/BRCA1 nuclear foci.

The FANCD2...

The Role Of Antibodies In Cobalt Chloride Induced Apoptosis In PC12 Cells

Monday, December 7, 2009 - 10:00

Various studies have been performed tracking the involvement of p38 mitogen-activated protein kinase (MAPK) and caspase-3 antibodies in cobalt chloride (CoCl2) induced apoptosis in PC12 cells.

Studies have proven the usefulness of CoCl2 treatment to induce apoptosis in PC12 rat tumor cells. Treating these cells with CoCl2 leads to programmed cell death (apoptosis). Such cells provide a simple and useful model for the study of hypoxia-related neuronal disorders, as hypoxia involves a lack of oxygen.

The mechanism of action is known to involve caspase cleavage. Antibody suppliers, such as Novus Biologicals, supply caspase and p38 antibody preparations, which are then used to study caspase cleavage in PC12 cells. p38 MAP Kinase (MAPK), also known as RK and CSBP, is known to be a pro-apoptotic factor, which when activated leads to the cleavage of caspase-3; thus, MAPK antibodies and...

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