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ANTIBODY CODE ab1162 BATCH NUMBER 50573 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE E.coli whole cell lysate containing DDDDK-tagged (internal tags) proteins PRIMARY ANTIBODY ab1162, AbCam anti-DDDDK rabbit IgG polyclonal 1/3000. Incubated 1h, washed 3x10min Tried Bethyl's version first, same type/conditions SECONDARY ANTIBODY Sigma goat anti-rabbit IgG-HRP conj. 1/3000, 1h incubation, 3x10min washes. DETECTION METHOD Western Lightning enhanced luminol/oxidising chemiluminescence reagents POSITIVE AND NEGATIVE CONTROLS USED +ve = enterokinase cleavage control protein included in recombinant EK kit from Novagen - contains DDDDK sequence. -ve = lysate of strain not containing expression plasmid for the DDDDK-tagged protein ANTIBODY STORAGE CONDITIONS 4 degC SAMPLE PREPARATION cell pellet resuspended and boiled 5 min in SDSPAGE loading buffer, reducing conditions, no protease inhibitors AMOUNT OF PROTEIN LOADED equivalent to 100ul of overnight culture, our protein is easily seen under the same conditions with a custom polyclonal specific for the protein of interest ELECTROPHORESIS/GEL CONDITIONS 10% SDS-PAGE non-reducing TRANSFER AND BLOCKING CONDITIONS semidry transfer, 20 min. Blocked O/N 4 degC with 5% skimmed milk in TBS-0.1% Tween + 5mMCaCl2 (previously tried 3% BSA in PBS-0.1%Tween) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? changed from original BSA/PBS-tween protocol which works well for our specific custom antibody to milk/TBS-tween with 2 then 5mM CaCl2 added to the TBS-Tween ADDITIONAL NOTES We originally got a ladder pattern on the western of non-specific bands, we were advised to use TBS in place of PBS and add CaCl2 in case Ca2+ in the system is sequestered by phophate - this removed the ladder pattern but we only got very weak signal (film down 40 min) with one of our DDDDK-tagged proteins +ve control did not come up so perhaps we should try the Invitrogen positope R900-40 as described in your datasheet? Any other recommendations for tweeking the binding would be much appreciated!
Asked on May 28 2004
Thank you very much for your patience. I'm sorry to hear that you are experiencing trouble with this antibody. At this point I would suggest increasing the concentration of the primary antibody and incubate with the primary for a longer period of time; the orginator of ab1162 incubated for at least 120 minutes and if needed, try incubating overnight at 4C. The originator has also suggested that perhaps you try the 12-tag cell lysate marker in your system. Also, you mentioned that you tried Bethyl's antibody first - did that work for you? If you continue to experience trouble with this antibody, please do let me know.
Answered on Jun 04 2004