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I am working with HeLa tumors, and with 1-D PAGE and WB: WB Amount of protein loaded : 40ug Blocking conditions : 5% Milk Detection method : Carbazol Electrophoresis and transfer conditions : 12% PAGE, Semi-dry transfer Primary antibody conditions (diluent/dilution, incubation time/temperature): TBST/1:10000,5hrs/24°C Secondary antibody conditions (diluent/dilution, incubation time/temperature): TSBT/1:10000,2hrs/24°C Wash steps: 20minutes X 3 times with TBST. As a positive control I use Mouse cerebrum. When I use 2D PAGE and Mass Spectometry with MALDI-TOF, I can identified the GSTM3 protein in the HeLa tumors, but, when I use the antibody in the WB I could not have signal, I want to know what peptide of the GSTM3 protein you have used to make the antibody, that way I can identified why I can identified GSTM3 with mass spectrometry but not with antibody. Any help would be welcome.
Asked on May 07 2013
The short peptide sequence that was used to raise this antibody is 14aa in length, 100% homologous to this protein (http://www.uniprot.org/uniprot/P21266) and contained within this larger 50aa sequence: EEKIRVDIIENQVMDFRTQLIRLCYSSDHEKLKPQYLEELPGQLKQFSMF. I would like to offer some suggestions to help optimize the results from ab74749. I see you have used this product at 1/10,000 dlution. We recommend a starting dilution of 1/1000 when using this in western blot and to incubate the antibody overnight at +4C.
Answered on May 07 2013