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LOT NUMBER GR43510-3 ORDER NUMBER 1007542 DESCRIPTION OF THE PROBLEM High background (LONP1 signal was so weakly.) SAMPLE - Species: human - What’s cell line or tissue: primary fibroblast cell (from the newborns) - Cell extract or Nuclear extract: cell extract - Purified protein or Recombinant protein: Whole cell extraction PRIMARY ANTIBODY - Species: rabbit polyclonal - Reacts against: human - At what dilution(s) have you tested this antibody: 1:1000 - What dilution buffer was used: 5% non-fat-milk in TBST - Incubation time: 1hr - Incubation temperature: room temperature - What washing steps were done: 15min x 1 + 5min x 3 DETECTION METHOD ECl+ ANTIBODY STORAGE CONDITIONS -20 SAMPLE PREPARATION - What lysis buffer was used: 20mM potassium phosphate buffer + sonication - What protease inhibitors were used: cocktail protease inhibitor - What loading buffer was used: NuPAGE LDS sample buffer (invitrogen:Cat no.NP0007) - Phosphatase inhibitors - Did you heat the samples: temperature and time: 70℃ for 10min AMOUNT OF PROTEIN LOADED 30ug ELECTROPHORESIS/GEL CONDITIONS - Reducing or non reducing gel: Reducing - Reducing agent: 2-ME - Gel percentage : 4-12% TRANSFER AND BLOCKING CONDITIONS - Transfer conditions: (Type of membrane, Protein transfer verified): PVDF, Coomassie blue (Invitrogen iBlot dry blotting system Cat no. IB10001) Blocking conditions - Buffer: 5% non-fat-milk in TBST - Blocking agent: milk, BSA, serum, what percentage: 5% - Incubation time: 1hr - Incubation temperature: room temperature SECONDARY ANTIBODY - Species: goat - Reacts against: rabbit - At what dilution(s) have you tested this antibody: 1:5000 - Incubation time: 1hr - Wash steps: 15min x 1 + 5min x 3 - Fluorochrome or enzyme conjugate: enzyme conjugate - Do you know whether the problems you are experiencing come from the secondary? HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No
Asked on Jan 16 2012
Thank you for your enquiry regarding ab103809 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody. Additionally, thank you for supplying an image, as this has been very beneficial in understanding your concerns.
The protocol looks fine to me however I would like to make few comments/suggestions that might help to improve your results and that you could consider trying:
The 100 kDa band presented on the online datasheet is with tissue lysates of human kidney, mouse and cell lysates of HepG2 cells. After reviewing the image you have kindly sent it looks like the ab is showing multiple bands. I would recommend troubleshooting the protocol further. My suggestions would be
- Not sure if phosphate buffer was used a lysis buffer? We recommend using NP40 buffer.
- When doing wb we heated the sample for 10 minutes at 95-100 C. Could you tty the same?
- We normally use 5% BSA in TBST (2 hrs 4C) for antibody dilution and blocking? I would recommend trying this.
- Try the above mentioned tissue lysates as positive control.
I am sure changing these steps will help to improve results. Should the results do not improve please do not hesitate to contact me.
Answered on Jan 16 2012