Alternatives to (weather forecast) 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.
The Article is written by novusbio.com/ providing antibody catalogue and antibodies Services. Visit http://www.novusbio.com/ for more information on novusbio.com/Products & Services___________________________Copyright information
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Markers and gels in Western blot immunoassays
By Rick Dahne
Western blot gel electrophoresis using antibodies is used in a range of biological research disciplines including molecular biology, immunogenetics, biochemistry and histology.
Typically, tissues are mechanically and chemically broken down to release cell proteins. Buffers, inhibitors and stabilisers are used to denature and stabilise these proteins, which are separated into individual target antigens each of which reacts to a specific antibody on a membrane.
The most common Western blot assay uses SDS-PAGE (sodium dodecyl sulphate/ polyacrylamide gel electrophoresis). Denatured proteins, negatively charged with a coating of SDS, are loaded into wells in the gel, one to each lane. One lane is reserved for a marker protein mixture, i.e. one with known molecular weights. When a current is applied, proteins migrate to a positive electrode through the polyacrylamide gel mesh. Smaller proteins, i.e. those with lower molecular weights migrate faster, thus the proteins form distinct bands which can be read off against the marker. The gel concentration can be tailored to achieve optimum resolution. For example, a higher concentration gel achieves better clarity with low weight proteins.
Western blot uses antibody detection to identify proteins. For this to be done, the proteins are moved onto a nitrocellulose or polyvinyl difluoride (PVDF) membrane PVDF being superior. Transference is achieved by placing a stack of filter papers on top of the membrane and using capillary action to draw the proteins off the gel. Electroblotting is an alternative method.
The membrane works by binding protein to its structure. Thus it must be blocked from interaction with the antibody proteins when they are applied. This is done by immersing the membrane in a bovine albumin or non-fat milk solution. The diluted globulins attach to all the places not occupied by the target proteins thereby preventing false positives.
We at Novus Biologicals have a vast antibody database, with many thousands of immunoglobulins suitable for Western blotting and other assays.
The Article is written by novusbio.com/ providing antibody database and antibody catalogue 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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