Friday, July 24, 2009

Monoclonal antibodies to Dicer - CG006 and CG016

The ribonuclease enzyme Dicer is an important cellular protein since it catalyses a rate limiting step in the production of microRNAs. Early experiments showed that disruption of Dicer led to a decrease in microRNA activity and significant effects upon cell growth and differentiation. In humans, germ line disruption of Dicer leads to Familial Pleuropulmonary Blastoma. Several studies have documented that expression of Dicer is related to the progression or grade of human tumors. Thus there is a need to develop reagents that will identify the Dicer protein. Clonegene has developed two monoclonal antibodies against Dicer the first CG006 has been selected by its ability to work in western blot analysis, although it will also perform moderately well in immunohistochemical and fluorescence studies. The second more recently developed antibody, CG016, has been selected for its ability to detect Dicer in human formalin fixed paraffin human sections on immunohistochemical analysis. It is important to note that it does also perform well in western blots. The specificity of both antibodies has been checked by siRNA directed depletion of Dicer. CG006 has been used in many of the pionering studies on the expression of Dicer in human tumors. The figure shows the reactivity of CG016 with FFPE sections of normal ovarian mucosa. CG016 and CG006 were generated against a conserved peptide antigen. Thus, reactivity has been seen with the closely related mouse Dicer protein. We cannot reveal the sequence of the peptide antigen used to make these antibodies but we are happy to let you know if they will likely react with Dicer from the organism that you are studying.

CG006 is available as a purified monoclonal is very heat stable and routinely shipped at room temperature in a proprietary stabilizer solution. But it is convenient to keep it in fridge for short-term use (weeks) or in freezer for long-term use (months). For immunochemistry or immunofluoresence we use 1/500 to 1/2000 dilutions and use standard anti mouse ABC peroxidase detection systems from Vector labs. For western blots we use similar dilutions and the standard anti – mouse systems using ECL detection.
Cg016 is currently available as a hybridoma and is also heat stable and shipped at room temperature. It is convenient to keep it in fridge for short-term use (weeks) or in freezer for long-term use (months). For detection of Dicer on paraffin sections, we use dilution of 1/50 to 1/200 and the vector labs ABC elite detection kit. The sections are treated with RETRIEVIA as discussed in earlier post.

Reblog this post [with Zemanta]

No comments: