Housekeeping protein issues - Is my experiment affecting my housekeeping protein levels? (Jun/29/2011 )
this is an unusual problem, but there may be someone out there who has been in a similar situation.
I have an experiment where I induce hypoxia on cell samples, collecting them at specific time points over 24 hours, and measuring a particular connexin level in them.
I measure the protein concentration of the samples using a Bradford Assay, to ensure that I have the same level of protein in all my lanes.
In my control samples (no hypoxia), my housekeeping antibody (alpha tubulin) levels are pretty similar over the 24 hour period, reassuring me that I do have similar levels of proteins in my lanes.
However, in my hypoxic samples, this is not the case. The levels of alpha tubulin rise progressively over the 24 hour time period, looking like I have not considered protein concentration over time.
Is there a slim possibilty that my house keeping antibody is being affected by the hypoxic nature of my experiment? I have heard that alpha tubulin is a pretty reliable house keeping antibody, so I don't know if this will be the case.
I have triple checked my calculations of my protein concentration (and will be doing another Bradford assay in the near future), but I really don't think that I have made a mistake here.
If anyone else has come across their housekeeping antibody being affected by their experiment, please could you throw some advice this way!
I guess one obvious option that I have is to change my housekeeping protein - but I don't want to run into the same problem.
Thanks in advance
ischamic and hypoxic situations are serious situations for cells and induce multiple processes; I think that it is possible to affect expression of so-called house-keeing genes; if you have the possibility check tubulin mRNA expression with PCR or take total protein amount as reference