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Although I wanted to use this antibody for human Heptoma cells (HepG2), I have tried to use this antibody using a variety of tissue samples (mouse heart, liver, kidney, primary hepatocytes as well as Human HepG2 cells). I am getting a lot of non-specific bands irrespective of the conditions/troubleshooting that I have done. Here are the conditions tried: Fresh tissue/cells were lysed and 10-100 ug of protein was loaded in reduced and denaturing conditions (DTT along with boiling at 95 deg C for 3 mins) in different experiments. In most experiments the whole cell lysate (cytosolic + membrane fractions together) was used, but in one experiment after cell lysis the supernatant and pellet (membrane) were ran separately. The gel was run at 30mA in a Tris-glycine based buffer with SDS and was transferred in a Tris-Glycine-Methanol based buffer under 100V for a little more than an hour. 3% BSA in TBS-T was used for blocking (both overnight at 4 deg C as well as 2 hrs at RT). Primary antibody was diluted in 1% BSA in TBS-T at specified concentration (as per product data sheet). Blot was incubated in primary antibody. Different incubation times were tried. 3hrs RT as well as overnight 4 deg C. Secondary body was Anti rabbit IgG. Both AP and HRP substrates were tried. Incubation time 1 hour and exposure time for SCL was upto 15 mins. I really need an antibody that works with my cells (HepG2 cells). Please help!!
Asked on Jun 14 2012
Thank you for sending your protocol and blot images for this antibody and for ab77612, Anti-Adiponectin Receptor 2.
I think your protocol is correct. Have you tried staining blots of these samples with any other antibodies using this protocol? My concern is about the quality of the samples. Were they harvested with protease inhibitors? If not, there is a possibility that they are degraded. However, you should still see a more prominent signal at the expected molecular weights.
Another consideration is how the samples are prepared before loading into the gel. Membrane receptors have a tendency to aggregate when boiled, and consequently do not enter the gel, so many researchers will heat samples to no hotter than 60-70C. This will denature the sample without causing the membrane proteins to aggregate.
If you are confident that your sample preparation is not the issue , then it may be best to replace the antibodies. Can you please tell me when you ordered them, and the order number?
Answered on Jun 14 2012