Description
The von Hippel-Lindau (VHL) protein is a tumor suppressor that is highly conserved from Drosophila to mammals. VHL protein normally associates with the transcription factor Elongin. Transcription factors typically act in one of three transcriptional stages, initiation, elongation or termination. Elongin functions by increasing the rate of elongation, the rate at which RNA subunits are added to make an RNA transcript longer. Elongin is made up of three subunits, A, B, and C, and each subunit contributes to the function of the whole protein. VHL binds to Elongin B and C subunits, and appears to prevent Elongin A from associating with the B and C subunits, thereby inhibiting the transcriptional activity of Elongin. The ability of VHL to block Elongin function may play a role in normal cell growth regulation. Mutated VHL sequences lose their ability to bind to Elongin subunits B and C, suggesting that the tumor suppression function of VHL may be linked to its ability to bind to Elongin. Likewise, the introduction of wild-type VHL suppresses growth in tumor cell lines lacking normal expression of VHL genes, further supporting a role for VHL in negative growth regulation. The calculated molecular weight of VHL is 24 kDa. However, different migrating species ranging from 21-30 kDa have been observed using antibodies to VHL, and may result from a variety of factors including alternatively spliced VHL mRNAs and protein degradation. This antibody has been reported to recognize both human and mouse VHL.
Western Blot: Clone Ig32 is not recommended for Western blot application. It has been shown to recognize a non-specific band above VHL protein. However, RNAi studies have demostrated that clone Ig32 recognizes a fainter band below this dominant band. Also, labs have reported using the 786-O renal cell adenocarcinoma cell line (ATCC CRL-1932, VHL null), siRNA or VHL overexpression to show that clone Ig32 recognizes the VHL protein specifically. Therefore, we recommend using clone Ig32 in over-expressing systems or under native (non-denaturing) conditions such as IHC staining or immunoprecipitation (IP) applications. Immunohistochemistry: The Ig32 antibody is useful for immunohistochemical staining. Following Retrievagen A pretreatment, purified Ig32 antibody should be used at 2.5 µg/ml to 5 µg/ml and titrated for optimal indirect immunohistochemical staining. Tissues can be visualized via a three-step staining procedure in combination with Biotin Goat anti-Mouse Ig (Cat. No. 550337) secondary antibody and Streptravidin-HRP (Cat. No. 550946) together with the DAB Substrate Kit (Cat. No. 550880). More conveniently, the Anti-Mouse Ig HRP Detection Kit (Cat. No. 551011) that contains the biotinylated secondary antibody, antibody diluent, streptavidin-HRP and DAB substrate can be used for staining. For IHC staining on paraffin sections, please refer to http://www.bdbiosciences.com/documents/Immuno_Prep_Stain_Paraffin.pdf Additional protocol information can be found at http://www.bdbiosciences.com/support/resources/cell_biology/index.jsp IP: The purified Ig32 antibody has been reported to be useful to immunoprecipitate native human VHL. Please note that this application is not routinely tested at BD Biosciences Pharmingen. Investigators are advised to determine optimal concentrations for individual applications.
Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
3. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
