Cholera Toxin, B Subunit (Choleragenoid) (HRP)

Cholera toxin is an oligomeric protein of MW 84kD and consists of a single A subunit surrounded by five B subunits. It is a potent activator of adenylate cyclase and is the pathogenic agent responsible for the symptoms of cholera. The B subunit (choleragenoid) is responsible for the binding of the holotoxin to G M1 ganglioside receptors on mammalian cell surfaces and facilitates entrance of the A subunit into the cell. The A subunit bears the ADP-ribosyl-transferase activity, which deregulates the G sprotein causing activation of adenylate cyclase. Due to the ubiquitous occurrence of the GM1 ganglioside receptor on eukaryotic cell membranes, cholera toxin activates adenylate cyclase in a wide variety of systems.\n\nCholera toxin has become a powerful research tool not only in microbiology, but in the fields of physiology, cell biology and biochemistry, as well. Because of the effect on adenylate cyclase, cholera toxin and its purified A subunit are frequently used for the study of signal transduction mechanisms. In addition, cholera toxin acts as an adjuvant through the stimulation of B-lymphocytes. The cholera toxin B subunit alone is used for track tracing in neurological research, taking advantage of GM1 ganglioside binding and retrograde transport.\n\nCholera toxin is isolated from Vibrio cholerae type Inaba 569B by modification of the methods of Rappaport et al. and Mekalanos et al. When equal weights are compared, the A subunit exhibits 3 to 5 times the transferase activity of the holotoxin. The B subunit exhibits little to no transferase activity.\n\nCholera toxin and native subunits all undergo treatment for the removal of contaminating endotoxin and are sterile as packaged.\n\nActivity:\nThe enzymatic activity in each preparation, is determined spectrophotometrically and reported ~200 purpurogallin units per mg of HRP.\n\nRecommended Dilution:\nPublished reports suggest that one-tenth to one-fiftieth the amount normally employed for free HRP would be an approximate starting range. If this product is compared to WGHRP, select several dosages in the range normally used with WGHRP. Keep in mind that the 0.1mg size of this product refers to the quantity of HRP per vial and not to total protein. Because this product diffuses less than other commercial preparations, multiple injection sites may be required. Projections to the amygdala have been successfully visualized following multiple injections of as little as 50ng of conjugate into rat brainstem, while 5ug of conjugate has been injected into rat tongue for visualization of cell bodies and dendrites in the hypoglossal nucleus.\n\nIncubation of Specimens:\nStrict adherence to the TMB incubation protocol as described by Mesulam et al. is strongly recommended with this product. Free floating sections are incubated in TMB for 10 minutes. This solution should then be replaced with a fresh TMB solution which contains the hydrogen peroxide.\n\nStorage and Stability:\nThis product has been stoppered under vacuum. When reconstituted with 100ul of distilled water, the solution will be 0.1% (1mg/ml) with respect to HRP. The conjugate may be reconstituted with less water if a more concentrated solution is required for injection. For example, to prepare a 1.0% (10mg/ml) solution, reconstitute with 10ul of water. Swirl gently and avoid excessive agitation. Store at 4 degrees C prior to and following reconstitution. Do not freeze.\n\nHandling:\nGood laboratory technique should be employed in the safe handling of this product. This requires observing the following practices: \n1. Wear appropriate laboratory attire including a lab coat, gloves and safety glasses. \n2. Do not mouth pipette, inhale, ingest or allow to come into contact with open wounds. Wash thoroughly any area of the body which comes into contact with the product. \n3. Avoid accidental autoinoculation by exercising extreme care when handling in conjunction with any injection device.

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SPECIFICATIONS

Catalog Number

C4275-36E

Size

100ug

Form

Supplied as a lyophilized powder in 0.01 M sodium phosphate, pH 7.5. No preservative added. Conjugated to horseradish peroxidase (HRP). Each vial of Cholera-HRP conjugate contains approximately 0.1mg of horseradish peroxidase (HRP) and approximately 0.048mg of the B subunit of cholera toxin as determined by extinction at 403 and 280nm, respectively.

Purity

The transferase activity of this lot was nearly zero when compared to a comparable amount of cholera toxin standard in an ADP-ribosylation assay. This preparation migrates as a single major band in disc gel electrophoresis under nondenaturing conditions. The R280/260 of each preparation is reported.

References

1. Mesulam, et al. (1980) J. Histochem. Cytochem. 28, 1255-1259. Finkelstein, R.A., Boesman, M., Neoh, S.H., LaRue, M.K. and Delaney, R. (1974) J. Immun. 113 ,145-150. 2. Gill, D.M. (1976) Biochemistry 15 , 1242-1248. Lai, C.Y. (1977) J. Biol. Chem. 252 , 7249-7256. Finkelstein, R.A. (1973) CRC Crit. Rev. Microbiol. 2, 553-623. 3. van Heyningen, W.E. (1974) Nature 249 , 415-417. 4. Holmgren, J. and L