Acid Bit

By Brigette Federico

 Immunoassays such as Western blotting and ELISA often rely on a two-stage technique. A primary antibody (1o Ab) is used to bind directly to the protein of interest. A secondary antibody (2o Ab), labelled with a detection tag such as a fluorescent dye or colourmetric enzyme, is then applied. This binds to a site on the primary Ab, allowing indirect detection of the antigen.

Single stage assays have been developed, in which primary immunoglobulins are tagged, allowing both probing and detection to take place from the same molecule. However, the indirect approach is still preferred, for many reasons. One-step 1o Abs are custom-built and thus expensive. In addition, more than one 2o Ab can target the primary protein producing clearer results.

Primary Abs are typically purchased from antibody suppliers in monoclonal form. These are derived from a single cell-line and are totally homogenous. By contrast, most secondary proteins are polyclonal. Here, animals are injected with a specific peptide antigen of a particular species, a secondary immune response is triggered and in this way a whole plethora of proteins are produced unique for that antigen.

The difference between monoclonal and polyclonal proteins is that, while a monoclonal only recognises one specific epitope, polyclonals can recognise several. The primaries are raised against a specific antigen, and are unlabelled. 2o Abs are raised against primaries, and are labelled, or conjugated, with a reporter enzyme or one of the new fluorescent products such as Dylight.

Sometimes best results are obtained by using antibody fragments, such as an Fab fragment or gamma light chain, as the primary Ab rather than the entire protein. This can improve signal-to-noise ratio in assays, and improve binding to the antigen. The 2o Ab will be specific to Fab.

The secondary antibodies supplied by us at Novus Biologicals are available for a wide range of specie and primary proteins.

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Studies using Bcl-2 antibodies

By Tom Jui

 The Bcl-2 gene family is important in regulating apoptosis, i.e. programmed cell death. The use of Bcl antibodies forms an important part of disease research at the cellular level. Apoptosis is essential for efficient cellular function and is utilised in many areas; for example homeostasis, defence against pathogens and embryonic development.

Bcl-2 proteins control apoptosis mainly by regulating the permeability of the outer mitochondrial membrane. Mitochondrial function depends on a switch-on/switch-off mechanism; therefore both pro and antipoptotic Bcl-2 proteins are involved. Homeostasis is achieved by a balance of functions between these two groups.

There are a large number of Bcl-2 proteins, each with a target antibody which is used for detection and quantitative analysis during immunoassays. Bcl-2 covers 4 BH domains. These domains regulate and control protein function and heterodimerisation between different Bcl-2 members.

Antipoptotic Bcl-2 proteins promote survival of the cell. These include A1, Mcl-1, 2Bcl-2, Bcl-2, Bcl-XL and Bcl-W. These contain all 4 domains. The multidomain proapoptotic (antisurvival) group of Bax, Bok and Bak contains BH domains 1, 2, and 3. The second proapoptotic group, BH3-only, comprises the Bad, Bid, Bik, Bim, Blk, Bmf, Bnip, Bcl-XS, Noxa, and Puma globulins. These have only the BH3 domain.

It is the ratio of pro to antisurvival Bcl-2 proteins which determine how susceptible cells are to apoptotic stimuli. Disruption of these levels, by increased or decreased expression of one or more proteins, is implicated in the development of various malignant tumours as well as non-malignant growths, AIDs, Downs Syndrome and diabetes.

Antipoptotic Bcl-2 is widely used in cancer research. Elevated levels are implicated in a number of tumours, and cause cellular resistance to proapoptotic proteins and chemotherapy treatments. On a more general scale, Bcl-2 proteins are useful biomarkers. Antibody suppliers like us at Novus Biologicals have a large range of marker protein antibodies on our databases.

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!

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