The detection of HuR in tumor tissues.

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. HuR is a nuclear protein and it's activation is signalled by its transport to the cytoplasm. The cytoplasmic localization of HuR has been corrrelated to the grade or prognosis of human tumors. Clonegene's CG001 monoclonal is the reagent of choice in determining the expresison of HuR in parafin embedded archival tumor sections. In the example shown to the right, CG001 was diluted 1/1000 and incubated with tumor sections for one hour at room temperature. (The section was deparafinized and treated with RETREVIA ). Bound antibody was revealed by incubation with standard anti-mouse secondary followed by Vector Labs ABc elite peroxidase reagents. For more information on CG001, contact Clonegene or email : info@clonegene.com

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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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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