Hello!
Currently I am testing whether an externally added agent affects the expression of only one particular protein or whether it generally inhibits protein synthesis due to its toxicity.
For this purpose, I add this agent to THP-1 monocytes and measure the concentration of MHCII proteins via Western Blotting as well as by flow cytometry after different time points (4h,8h,16h,24h).
I observe a clear time-dependent reduction of the MHCII levels.
However, I need suitable controls to find out whether the effect of this agent is specific for MHCII expression or whether it generally decreases protein synthesis. I suppose that GAP-DH is too stable and thus, the levels will likely remain stable during the chosen incubation time irrespective of the presence of the agent.
Do you know other proteins which are short-lived but continuously expressed and thus, present at nearly constant levels?
Thanks for your input!
Hi,
This research article will be helpful for your study. In this research article, they mentioned some other housekeeping genes that may be significant for your experiments.
Article Selection of reliable reference genes for the normalisation ...
This strongly dependyx on your experimental setting (e.g. time, stimulus, media etc.
If GAPDH is too stable, beta-actin could be an alternative. In general, structure-related proteins should be continously expressed.
Hello Agnes,
Choosing a stable reference protein for your experiment is largely dependent on your treatment, as what Andreas mentioned; from a treatment to another those proteins may vary in their expression.
B-Actin and GAPDH are the most used loading control targets in such experiments due to their usually stable and ubiquitous expression.
On the other hand, i advise you to check the following articles on another interesting technique that may solve your problem in western blot normalisation:
Article Ponceau S waste: Ponceau S staining for total protein normalization
Article Total protein or high-abundance protein: Which offers the be...
Article Use of the REVERT ® total protein stain as a loading control...
Chapter Normalized Quantitative Western Blotting Based on Standardiz...
This technique uses the total protein rather than a high-abundance protein (GAPDH, Etc.) to normalise your samples.
Hopefully my answer has been helpful for you,
Sincerely,
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