I have done my western blotting using C2C12 cell lysate sample but I am not able to get the band for the above.
I am using Santa Cruz antibodies for the insulin receptor beta subunit and its phosphorylated form.
Details?
Are you running gels with or without the SDS?
The C2C12 lysate is supplied in buffer that consists nonionic and ionic detergents, including 0.2% SDS. If you are running denatured gels, addition of one or a few phosphates isn't likely to change the molecular weight. Try a gel without SDS, charge difference will separate unphosphorylated forn from phosphorylated version quite nicely. A better but slightly tedious method is to perform a 2-D gel.
If you can find an antibody that specifically recognized phosphorylated InsR, that would be helpful.
Hi Revathy, I donn't known very well the insulin pathway, but, even if Neus point out an important "technical" aspect (your antibodies are new, they were used by someone else ?), I think tha you have also to check the basic expression of this receptor from literature. Did the receptor is normally expressed in C2C12? Is it expressed in myoblast and in myotube? How much is expressed, maybe you have to load a lot more lysate? For the phosphorylated one, I think you treated the cells (for example ith insulin), did you think that the experiment (treatment) work well? Maybe the treatment failed, for this reaon you din't see the phospho-receptor? If you can exclude technical problems read some literature to understand why you don't see anything.
thank you people.
but I am able to get a bamd fror the downstream proteins except IR and IR beta following stimulation with insulin.
Hi, you already verify that your stimulation work well, the only thing that it is strange is that you don't have the signal not only of phopshorylated receptor but also of non-phopshorylated one. If the Santacruz datasheet assure you the detection in mouse cell lysates (with an image), you can contact the Santacruz support desk, they help me with an antibody (at the end was so old that I have to buy it again).
You try with super ECL only to confirm that you didn't reveal anything?
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