I used Ser 307 IRS antibody at a concentration of 1:1000 and it was purchased from CST as IRS antibody sampler kit.
Following the manufacturer's recommendations is always a good starting point.
Use as much protein as you can for your samples and include a positive control.
Can you detect IRS-1 itself in the non-phospho form? If not - you will need to alter your transfer settings (perhaps less methanol, longer transfer time, pvdf membrane, ect...). Do you have phosphatase inhibitors in your extraction buffer? If you can detect the total protein and you have phos inhibitors in your extraction buffer, I would suggest going to 1:500 (overnight in 4 degree fridge) and adding phos inhibitors during the western blot - as per Sharma and Carew 2002 (see attached).
If the reagents are available, you could also try flow cytometric analysis or ELISA. these are more sensitive. Good luck.
I used 1:500 dilution and loaded most probably 50 ug protein. But the signal was not always good and so finally I did it by Elisa.
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