Hello Aleena Varughese

Housekeeping gene is essential for cellular existence regardless of their specific function in the tissue or organism, and it is stably expressed irrespective of tissue type, developmental stage, cell cycle state, or external signal.

The housekeeping gene that you select for your experiment, should show no or only minimal changes in expression levels between the individual samples and under different experimental conditions.

β-actin is commonly used as a housekeeping gene for RT-PCR and Western Blot because it is regarded as a highly stable housekeeping gene. But this may not always be true! Sometimes, the expression of β-actin may significantly change making it difficult to be used as suitable internal control as it may cause problems in data acquisition, analysis, and interpretation. In such cases, you will have to look out for another housekeeping gene that shows stable expression for your type of experiment.

The best way to select the most appropriate housekeeping gene/protein for a qPCR/Western Blot experiment would be to select some candidates and determine their expression levels across the range of experimental conditions and treatments. Those candidates that are most stably expressed across these conditions would be the appropriate housekeeping gene/protein for your experiment. It is not just β-actin, you could also select from other commonly used housekeeping genes such as GAPDH, β-tubulin or β-2-microglobulin.

There is nothing such as normal range for cell lines. It can vary depending on the experimental conditions. The important thing to be noted is that the Ct value of the housekeeping gene should be stable across different samples and under different treatment conditions.

Best.

As a small addition to what was mentioned. You have to take in your consideration that in WB, HKGs give a high rise to false interpretation in case you did not validate them.

Regarding RT-qPCR, selection of the appropriate HKG is challenging and you have to use at least two validated HKGs for normalization in triplicate. The normal Ct ranges in for HKGs is around 16.

Best

There is no range really besides the limit of reasonable quantitation. You generally strive for mid-high expression that is reproducible. For PCR that would probably mean you don't want something past 30-35 Cts, for Westerns you'd want something that transfers reasonably well, antibodies are specific and gives good signal. With Western blots, you may even consider proteins that are in the fraction of interest (mitochondrial, cytosol, membrane etc) as shown here https://www.abcam.com/primary-antibodies/loading-control-guide.

As Malcolm suggests, never assume that the common reference genes/proteins cited in the literature will be stably expressed in your conditions. I have often found this to be not true for GAPDH and Beta-actin during my doctorate studies. One additional nuance for Westerns is "The signal detected for both the loading control and the protein of interest must be in a linear range." as stated in https://blog.cellsignal.com/choosing-a-western-blot-loading-control-cst-blog.