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Our Abpromise guarantee covers the use of ab34726 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ELISA||1/10000 - 1/50000.|
|WB||1/500 - 1/3000. Detects a band of approximately 32 kDa (predicted molecular weight: 31 kDa).|
|IHC-P||Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
Fixation with PFA requires a denaturing step, see Abreview by M.Schrader.
ab34726 at a 1/100 dilution staining VDAC1/Porin in COS7 cells by Immunocytochemistry/ Immunofluorescence.The antibody does not work with a standard fixation for ICC/IF as it does not recognize the non-denatured protein. However, applying a fixation for hidden antigens (modified from Peränen J, Rikkonen M, Kääriäinen L. A method for exposing hidden antigenic sites in paraformaldehyde-fixed cultured cells, applied to initially unreactive antibodies. J Histochem Cytochem. 1993 Mar41(3):447-54) results in very good recognition of VDAC/porin. Denaturing is achieved by Guanidine hydrochloride in 50 mM Tris-HCL pH 7.5 after fixation (3% pFA, 0.05% GA) and permeabilization (0.2% TX-100). The secondary used was a TRITC conjugated donkey anti-rabbit used at a 1/100 dilution.