Before doing western we always denature proteins in 95 degree for 5 mins. My question is for example: cyclin E always forms complex together will cdk2 in its functional form. So after 95 degree 5 mins denature does the connection between cyclin E and cdk2 will be break or not? if we detect cyclin E with its specific antibody. Will we detect cyclin E at 48kDa position or is that possible that we could dectect the cyclin E/cdk2 complex at 48+33 kDa?
Thank you all in advance!
Only single blot at 48kDa is correct. Cdk2 will be checked separately using cdk2 specific Ab. Thanks
Heating as well as breaking bonds between proteins chemically are critical for detection of proteins that are often found in protein complexes. Therefore, if one is using Laemmli Buffer with a reducing agent (DTT or Beta-ME) to heat proteins at the temperature you indicated, it is likely that one would detect cyclin E as a 48 kDa protein.
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