Western blotting with an anti-HuR monoclonal

The HuR protein is an important regulator of gene expression. It has been shown in many studies that HuR mediates the effects of cellular signals that upregulate gene expression at theRNA level. In general, such signaling pathways lead to an increased binding of HuR to particular elements within the mRNA, which abrogates the effect of factors that degrade mRNA. More recently, HuR has been identified as an important cofactor in the action of microRNAs. Clonegene has developed high affinity monoclonal antibodies to HuR that have been used in many of these studies. CG001 works well in western blot analysis. CG001 is supplied as a purified monoclonal. For westerns, use at 1/1000 ( 2 hours incubation at RT ) or 1/5000 ( overnight in cold room) and a standard anti-mouse secondary coupled to ECL detection. The figure show the reactivity of CG001 with extracts ( 50ug protein per lane ) from Hela cells. Lane 1 is no antibody , lane 2 is CG001 , lane 3 CG001 competed with antigen peptide , lane 4 CG001 competed with irrelevant peptide and lane 5 irrelevant antibody. The apparent molecular weight of HuR is 36 kD.
For further information contact clongene or email info@clonegene.com.

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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
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
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.

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RETREVIA - A new antigen retrieval reagent for IHC

Pathology samples are routinely fixed in formaldehyde, embedded in paraffin blocks (FFPE), cut into sections, stained with dyes and viewed under a microscope. This method preserves cellular structure and reveals considerable detail. Such blocks are collected over many years and thus provide an invaluable clinical database for studies on gene expression. This method is of particular utility in studies of protein expression in human tumor surgical samples. There are now many examples where the detection of particular proteins directs a specific therapy. For example the overexpression of HER-2 prompts treatment with Trastuzumab
However, this fixation method cross-links proteins and thus these sections do not work well in standard immunohistochemical assays. In most cases the epitope recognized by a particular the cross-linked residues obscure antibody and little reactivity is possible. To reactivate the protein antigen, the sections are heated in a buffer, which removes the crosslinks. Some protocols recommend steam heat some recommend microwave. Clonegene has now launched a new retrieval reagent called RETREVIA. The above picture shows the strong reactivity of a lung cancer section with our anti-HuR antibody (CG001) after treatment with RETREVIA .
Below we summarize the key steps that we recommend to give superior staining with our antibodies
1. Deparaffinize sections in 2 changes of xylene, 5 minutes each.
2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each. Then rinse in distilled water.
3. Pre-heat steamer or water bath with staining dish containing RETREVIA (dilute 10 ml of RETREVIA to 1 Litre of distilled water) until temperature reaches 95-100 °C.
4. Immerse slides in the staining dish. Place the lid loosely on the staining dish and incubate for 30 minutes (however this incubation time may depend on the nature of the sections and should be determined by user).
5. Turn off steamer or water bath and remove the staining dishes to room temperature and allow the slides to cool for 20 minutes.
6. Rinse sections in PBS for 2x2 min.

Proceed to standard immunohistochemistry protocol, we recommend Vectors labs ABC elite staining kit.
RETREVIA also works well in automated staining platforms.

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