(Weather forecast) Alternatives to embryonic stem-cell lines in antibody research
No commentsBy Davis Morris
Many suppliers include a range of stem cell marker proteins on their antibody databases. Marker antibodies, such as the one selective for Podocalycin-like protein (PODXL) are widely used in research into cancers and other diseases.
The PODXL gene has a similar structure to the CD-34 ligand, and encodes to a member of the sialomucin protein group. Originally identified on epithelial podocyte cells, it has been implicated in the development of aggressive tumours, and is used as a diagnostic marker in numerous cancers, including prostate cancer and pancreatic carcinomas.
At Novus Biologicals, our PODXL TRA-1-81antibody is one of a number of cell surface marker immunoglobulins designed for embryonic stem cell research. It recognises a specific carbohydrate epitope on the PODXL protein, the antigen TRA-1-81. Involved in cell differentiation, TRA-1-81 is expressed through PODXL on the surface of embryonic stem and germ cells, and also upon the surface of adult human teratocarcinoma stem cells. Cell surface antigens are known to play an important role in the ex-pression and function of developmentally controlled cells during embryonic and tumour development. For this reason, embryonic stem cells are routinely used in cancer studies involving TRA-1-81 and other PODXL immunoglobulins.
Since no murine (mouse) immunoreactivity is seen with our TRA PODXL antibodies, human cells must be used. The area of human embryonic stem-cell (hES) research is an extremely controversial one. However, in January 2010 Mizrak, Chikhovskaya et al, of the University of Amsterdam, released a paper suggesting an alternative human adult tissue. Studies had already shown that multipotent ES-type cells could be sourced from adult mouse testes. When these experiments were repeated using tissue donated from prostate cancer patients, subcultured cells expressed hES specific carbohydrate antigens as in the mouse studies. These included TRA-1-60 and TRA-1-81 both of which are on our antibody database.
This study means that human stem cell research is no longer restricted to embryonic cell lines an exciting development in an area which is historically extremely controversial.
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Modern developments in Western blot techniques
By Gen Federico
Western blotting is a routine procedure in immunoassays. Proteins are first separated by gel electrophoresis, before being blotted onto a specialised membrane which binds with the protein. The membrane is blocked from further protein interaction, and is then incubated with antibodies targeted to the relevant antigens. These bind direct to the protein, hopefully with minimal interference from the substrate, producing a readable result.
Traditionally, protein detection has been a two-step, indirect process. The membrane-bound protein is incubated with a primary antibody, which is unlabelled and reacts with the antigen direct. The excess is then washed off and a secondary immunoglobulin applied, which binds to a certain site on the primary agent (i.e. an amino acid sequence). This is species-specific, and must be raised against the immunoblobulin of the species of the primary Ig. Secondary proteins are labelled (conjugated) with an enzyme or luminescent dye which, when activated, gives a readable signal.
Traditionally, a biotin-labelled secondary immunoglobulin is tagged with an enzyme such as horseradish peroxidise. This results in a reaction called DAB-staining which makes the proteins visible. They are then read in a spectrophotometer. Recently, fluorescent dyes such as DyLight have become a popular alternative. Here, the antibody is conjugated with a fluorescent probe which, when excited, fluoresces in the near-infra red range. Fluors are considered the most sensitive detection agents in blotting assays.
The two-step assay method remains popular because signals can be strongly amplified by using several secondary Igs, each of which bind to a different site on the primary structure. However, with improved detection methods and higher throughput techniques, interest has switched to one-step probing methods. This requires antibodies that recognise the target protein and are labelled for detectable protein tags.
We at Novus Biologicals are constantly striving to expand and improve our antibody database. Our labs produce a huge range of products, including the latest epitope tagged immunoglobulins.
The Article is written by novusbio.com/ providing antibody database and antibody Services. Visit http://www.novusbio.com/ for more information on novusbio.com/Products & Services___________________________Copyright information
This article is free for reproduction but must be reproduced in its entirety, including live links & this copyright statement must be included. Visit novusbio.com/ for more services!
The role of apoptosis antibodies in atherosclerosis research
By Arthor Greenwald
Apoptosis, or programmed cell death, is a beneficial and essential part of tissue growth. However, it is also implicated in certain diseases, and thus apoptosis proteins are heavily featured in the databases of antibody suppliers, such as us at Novus Biologicals.
Apoptosis is known to be triggered by cellular damage. Various proteins are implicated, depending on the cause. For example, apoptosis due to ionising radiation or chemotherapy is triggered via the p53 pathway. Cells expressing TNF, or tumour necrosis factor proteins can initiate apoptosis following cross-linking. Activation of caspase is another common cause.
Inhibition of apoptosis is important in certain cancers. For example, Bcl-2, an apoptosis blocker, is expressed in some leukaemias. Melanomas can inhibit expression of Apaf-1, which triggers apoptosis. However, cancer is not the only disease of interest; it is now thought it could lead to formation of atherosclerotic plaques as well. Studies have shown increased endothelial, macrophage and vascular smooth muscle cell apoptosis may initiate the disease and form lesions. However, the process of activation is still poorly understood.
Apoptosis involves a complex family of proteins, some of which inhibit and some of which activate cell death. Both types are implicated in disease. Recent studies have focussed on how these proteins relate to the formation of atherosclerotic lesions, using antibody assays to map the findings. Diseased artery sections from atherectomy patients were compared against non-diseased tissues. The degree of apoptosis was determined in each using TUNEL (TdT/dUTP nick end labelling) techniques. In addition, expression of apoptosis protein regulators using antibodies was performed via Western blotting and paraffin embedding.
The results showed that in TUNEL apoptotic samples, Bax and Bak were the only proteins showing a distinct presence, although the Bcl-2 antibody gave a positive trace in some cells. Bcl-x was absent from apoptotic cells, with only the long isoform, Bcl-xL present in controls. The conclusion was that increased Bax and Bak, combined with lack of Bcl-2 and Bcl-xL, is associated with lesion formation, with Bcl-xL appearing to contribute to lengthened cell survival in non-apoptotic cells.
The Article is written by novusbio.com/ providing antibody database and antibodies Services. Visit http://www.novusbio.com/ for more information on novusbio.com/Products & Services___________________________Copyright information
This article is free for reproduction but must be reproduced in its entirety, including live links & this copyright statement must be included. Visit novusbio.com/ for more services!
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